Download The Hype on the Endothelin Signaling System Muscarinic Receptor

Issue No.3 Winter 2010
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The Hype on the Endothelin Signaling System
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Colocalization of P2Y2 using Anti-P2Y2 antibody (#APR-010) and CYTK-20 in cat urothelium. DAPI is used as
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Molecular Tools for the Life Science Community
The Hype on the Endothelin Signaling System
Melanie Grably, Ph.D.
Over the past two decades, research in the endothelin signaling system has been
quite intensive since they are profoundly involved in hypertension and cardiovascular
diseases. While major efforts are being invested in finding a proper antagonist to the two
G-protein coupled receptors of endothelin, ET-A and ET-B, we provide a brief report on
advances made in the field of the endothelin receptors and how Alomone Labs products
have influenced the advances made.
Endothelins and Hypertension
Endothelin, a 21–amino acid peptide, has potent
and sustained vasoconstrictive effects on
vascular smooth muscle both in vivo and in vitro
and is found in three different isoforms; ET-1, ET-2
and ET-3. Although expressed by other cell types,
ET-1 is mainly expressed by the vascular system
in endothelial cells. ET-1 binds and activates
two G-protein coupled receptors (GPCRs), ET-A
and ET-B (extensively reviewed in Reference
1). While the former has been attributed to
vasoconstriction, the role of ET-B depends mostly
on its location; on one hand it could promote
vasoconstriction and on the other stimulate nitric
oxide (NO) release -a known vasodilator2. ET-1 is
implicated in pulmonary arterial hypertension as
well as systemic hypertension models including
deoxycorticosterone acetate-salt (DOCA)
hypertension3-5. Contributing factors to the
development of pulmonary hypertension are the
elevated levels of circulating endothelin (ET-1).
The direct implication of increased ET-1 on the
pulmonary circulation was studied4. The study
leads to the conclusion that following four weeks
of chronic administration of ET-1, pulmonary
hypertension was not developed. What was
observed however, among other effects, following
a 7-day administration of ET-1, was a three-fold
increase of ET-B (but not ET-A) in both the intima
and media of small pulmonary arteries detected
under confocal microscopy imaging using AntiET-A and Anti-ET-B antibodies ((#AER-001 and
#AER-002), respectively) which returned to normal
after 28 days (Figure 1). Surprisingly, there was no
increase in ET-1 expression in pulmonary arteries
which was explained by the assumption that
increased plasma ET-1 had no in vivo effect on
vascular smooth muscle ET-1 production4 (Figure
1).
Chronic intermittent hypoxia (CIH) is characterized
by very short hypoxic episodes followed by
2
Figure 1. Immunofluorescence Quantification of the Distribution of ET-A,
ET-B and ET-1 in Small Pulmonary Arteries.
ETA receptor
ETB receptor
ET-1
M
IEL
E
EEL
The images represent examples of composite Z stacks that were deconvolved and projected with the LSM 510 software (Zeiss).
The first row of figures displays fluorescence for the ET-A and ET-B receptors, using Anti-ET-A and Anti-ET-B antibodies (#AER001 and #AER-002) respectively and for ET-1, from left to right (in red). ET-B and not ET-A is detected in the endothelium (E).
The second row displays the same components, but with the addition of the internal elastic lamina (IEL) and external elastic
lamina (EEL; in green), which enables easy demarcation of the endothelium (E) from the media (M). The third row displays
the fluorescence for smooth muscle actin (in blue), which is limited to the media (middle image) and colocalizes with ET-A
(on the left). A phase contrast image is shown in the bottom right corner. Scale bar, 10 μm. The computed mean fluorescence
intensities are presented in the bar graphs. * P < 0.01 versus control 7 days.
Adapted from reference 4 with permission of the American Thoracic Society.
GPCR Pathways No.3 Winter 2010 www.alomone.com
normoxia, and is the main distinguishing
feature in sleep apnea which has been linked to
hypertension in humans6. Some of the effects
of CIH seem to occur in the carotid body (CB)
where a 10-fold increase in ET-1 has been found
in cats treated with CIH7. In the same study, the
increased discharges observed in the carotid
body of CIH-treated cats were decreased upon
treatment with pharmacological blockers of ET-A
and ET-B. Indeed, western blot analysis shows
that ET-B is present in the carotid body and that
its expression is increased in response to CIH.
Immunohistological studies also show similar
findings; that both receptors are present in
glomus cells of the carotid body8 (Figure 2).
Endothelins and Vascular
Dysfunctions
The involvement of the endothelin signaling
system is quite prominent and very much related
to diabetes complications leading to vascular
dysfunction in human and diabetic animal
models9-12. Ca2+ channel blockers are clinically
widely used for the management of hypertension,
angina pectoris, and cardiac arrhythmias13. The
long term effects of benidipine (a Ca2+ channel
blocker) on the endothelin system were studied
in a rat model of human Type 2 diabetes14. This
model shows an increase in ET-1 expression in
plasma and left ventricular (LV) tissues compared
with the non-diabetic controls. The diabetic
rat model also shows increased levels of the
ET-A and ET-B receptors as demonstrated by
immunohistological studies as well as western
blot analysis using Alomone Labs' Anti-ET-A and
Anti-ET-B (Figures 3A and B). Upon an 8 week
treatment with benidipine, ET-1, ET-A and ET-B
levels were similar to non-diabetic controls. In
addition, benidipine treatment strongly prevented
cardiomyocyte hypertrophy, partly attributed to
the decrease in expression levels of ET-1 and its
related molecules14.
Endothelins and the Renal
System
The endothelin receptor system is distinctly
involved in the regulation of renal function under
both physiological and pathophysiological
conditions. The effects of ET-1 in kidney have
been attributed to both subtypes of GPCRs by
the use of specific agonists and antagonists
of these receptors. The receptors also have
functions leading to vasoconstriction as well
as vasodilation, important in the regulation of
renal vascular resistance15,16. However, direct
evidence for the cellular distribution of both
receptors was limited until recently. Using AntiET-A, immunohistochemistry studies showed
that ET-A colocalizes with desmin in mesangial
cells17 (Figure 4A). In the renal cortex, ET-A also
is found to colocalize with aquaporin 2 (using
Anti-Aquaporin 2 antibody (#AQP-002)) in
collecting ducts but not in the outer medulla. In
order to indisputably show that the signals were
Expression of ET-B in Rat Thalamus.
Immunohistochemical staining of rat dorsal peri-ventricular
thalamus using Anti-ET-B antibody (#AER-002). Cresyl violet
is used as the counterstained. The specific staining is black.
Western blot analysis of rat brain
membranes:
1. Anti-ET-B antibody (#AER-002),
(1:200).
2. Anti-ET-B antibody, preincubated
with the control peptide antigen.
Expression of ET-A in Rat Cerebellum.
A
Figure 2. Immunohistochemistry of ET-A and ET-B Receptors in the Carotid Body.
B
Immunohistochemical staining of rat cerebellum using
Anti-ET-A antibody (#AER-001) (A), or Anti-ET-A antibody
preincubated with the control peptide antigen (B). Cresyl
violet is used as the counterstained. The specific staining
is black.
A) Using Anti-ET-A antibody (#AER-001), ET-A immunoreactivity is located in cells within the carotid body
lobules; these cells are also positive for TH (inset). B) Positive ET-A staining in arterioles and venules in
the carotid body vascular pole. C) and D) ET-B immunoreactivity using Anti-ET-B antibody (#AER-002), was
shown in the glomus cell cytoplasm, endothelium of the carotid body parenchymatous capillaries and
Western blot analysis of rat brain
venules. E) ET-B immunoreactivity was also shown in arteries, endothelium and tunica media of the vessels
membranes:
in the vascular pole. ET-B immunoreactivity was more intense in glomus cells of the CIH-exposed carotid
1. Anti-ET-A antibody (#AER-001),
bodies (D) compared with control carotid bodies (C). F) and G) Negative controls preabsorbed with control
(1:200).
peptides showed no staining. Scale bar, 20 μm.
2. Anti-ET-A antibody, preincubated
Adapted from reference 8 with permission of the Histochemical Society.
with the control peptide antigen.
GPCR Pathways No.3 Winter 2010 www.alomone.com
3
specific to the receptor, the immnunoreactivity
of the ET-A receptor was completely abolished by
preabsorbing Anti-ET-A with the antigen used for
immunization17 (Figure 4B).
The dopaminergic system also controls the renal
system. In a recent paper, it was shown that D3
dopamine receptors work in close concert with
ET-B in maintaining proper Na+ homeostasis in the
kidney. In fact, these two receptors colocalize in
immunohistochemistry studies18 (Figure 5).
Figure 3. ET-A and ET-B Levels Decrease in Left Ventricular Tissues upon
Benidipine Treatment.
Endothelins, Retina and Neurons
In most cases, photoreceptor injury leads to the
activation of Muller cells in the retina, where
the accumulation of glial fibrillary acidic protein
(GFAP) is observed. Although this type of cellular
response has probably evolved to limit neuronal
damage, excessive activation of Muller cells can
impair recovery19,20. The activation of Muller cells
in response to diverse retinal disorders suggests
that these cells are at the health checkpoint of
retinal neurons and perhaps do so by sensing
a neuron-derived signaling molecule released
by damaged or healthy neurons21. Briefly, this
possibility was assessed by using mouse retina
subjected by a severe light insult (consequently
leading to retinal degeneration) and monitoring
changes in gene expression21. Microarray
analysis was performed on retinas damaged by
acute exposure to visible light (known to also
lead to photoreceptor death). Genes that were
upregulated include ET-2, which accumulated in
photoreceptors, and ET-B which accumulated in
the inner nuclear layer (INL) where Muller cells are
normally distributed. Immunostaining using AntiET-B and anti-GFAP showed that the localization
of these two proteins overlapped (Figure 6). These
observations suggest that ET-2 may function as a
stress signal by activating ET-B located on Muller
cells, thereby activating these cells21. In contrast,
ET-1 seems to be the activator of both ET-A and
ET-B receptors (found in bipolar dendrites and
horizontal cells, using Anti-ET-A and Anti-ETB
respectively) following a non-acute light insult,
where a similar cell survival mechanism takes
place22.
An additional indication that the endothelin
signaling system is present and functional in the
neuronal system is from a recent study where
ischemia was inflicted in mice and ET-B presence
was monitored among various other parameters.
ET-B colocalized with GFAP and vimentin around
the ischemic lesion in immunohistological
studies while no receptor was detected in GFAP -/Vim -/- mice suggesting that the intermediate
filaments, GFAP and vimentin, are required for
the production, stability, or distribution of ET-B in
reactive astrocytes23 (Figure 7). In addition, using
cultured astrocytes the same study showed that
by laser-scanning confocal microscopy, there was
a remarkable colocalization between bundles of
intermediate filaments and ET-B immunoreactivity
in wild type cells23 (Figure 8).
4
A
i
ii
iii
B
A) Confocal images showing showing immunofluorescence labeling for ET-1 (i), ET-A (ii) and ET-B (iii) in left
ventricular tissue sections from control rats (LETO), diabetic rats (OLETF) and benidipine treated diabetic rats
(BEN) using Anti-ET-A and Anti-ET-B antibodies respectively (#AER-001 and #AER-002). Both vascular and
extravascular regions are more immunoreactive in diabetic rats than in those in control rats and Benidipinetreated rats. Original magnification is x400.
B) Western blot analysis of ET-A (left) and ET-B (right) from left ventricular tissues of control, diabetic and
benidipine-treated rats. ± SE; n = 12 animals. **P < 0.01 and ***P < 0.001, compared with corresponding values
obtained in LETO rats; ##P < 0.01, compared with corresponding values in OLETF rats without any treatment.
Adapted from reference 14 with permission of the American Physiological Society.
GPCR Pathways No.3 Winter 2010 www.alomone.com
Figure 4. Colocalization of ET-A and Desmin in Mesangial Cells.
A
B
A) Using Anti-ET-A antibody (#AER-001), ET-A receptor immunoreactivity
colocalizes with desmin on extra- and intraglomerular mesangial cells
as evidenced by the yellow color in the overlay picture. B) Preabsorbed
with the peptide used for immunization, no ET-A immunoreactivity can be
observed (right panel). CCD, cortical collecting ducts; Gl, glomerulus.
Adapted from reference 17 with permission of the Histochemical Society.
Figure 5. Colocalization of ET-B and D3 Dopamine Receptor in Rat Kidneys.
D3 dopamine receptor and ET-B colocalize using Anti-ET-B antibody
(#AER-002) in kidneys from rat. Colocalization appears as yellow after
merging the images of fluorescein isothiocyanate-tagged ET-B receptor
(green) and Alexa 568-tagged D3 receptor (red). No staining is seen
without the antibodies (DIC). DIC, differential interference contrast.
Adapted from reference 18 with permission of Nature Publishing Group.
Figure 6. Immunostaining of Accumulation of ET-B and GFAP in Muller Cells in Light-Damaged Retinas.
A)-F) Mice were exposed to 6 hours of bright light with pupil dilation, followed by a 24 hour recovery in darkness.
A)-C) Enlarged views of the central retina from D)-F). G)-I) Control mice maintained in darkness. Light exposure
leads to the accumulation of ET-B as shown using Anti-ET-B antibody (#AER-002) and anti-GFAP in Muller cells,
characterized in the central retina by their radial fibers. ET-B also accumulates in presumptive astrocytes in the GCL
and at the outer tips of the Muller cells, which lie alongside the photoreceptor inner segments between the outer
nuclear layer and the RPE. Among Muller cells in the light-damaged retina, both ET-B and GFAP show substantial
cell-to-cell variability. C, Choroid; ONL, outer nuclear layer; OPL, outer plexiform layer; IPL, inner plexiform layer.
Adapted from reference 21 with permission of The Society for Neuroscience.
GPCR Pathways No.3 Winter 2010 www.alomone.com
5
Figure 7. ET-B Immunoreactivity in Astrocytes Around the Ischemic Lesion in
WT but not GFAP -/-Vim -/- (GV) Mice.
References
1. Rubany, G.M. and Polokoff, M.A. (1994) Pharmacol. Rev. 46, 325.
2. Tykocki, N.R. et al. (2009) J. Pharmacol. Exp. Ther. 329, 875.
3. Dai, X. et al. (2004) Hypertension 43, 1048.
4. Migneault, A. et al. (2005) Am. J. Respir. Crit. Care Med. 171, 506.
5. Sauvageau, S. et al. (2006) Exp. Biol. Med. 231, 840.
6. Quan, S.F. and Gersh, B.J. (2004) Circulation 109, 951.
7. Rey, S. et al. (2006) Brain. Res. 1086, 152.
8. Rey, D. et al. (2007) J. Histochem. Cytochem. 55, 167.
9. Ferri, C. et al. (1997) Diabetologia 40, 100.
10. Piatti, P. et al. (1996) Diabetes 45, 316.
11. Sachidanandam, K. et al. (2008) Am. J. Physiol. 294, H2743.
12. Sachidanandam, K. et al. (2009) Am. J. Physiol. 296, R952.
13. Roberts, R. and Zanchetti, A. (1996) Cardiovascular Drug Therapy.
14. Jesmin, S. et al. (2006) Am. J. physiol. 290, H2146.
15. Lanese, D.M. and Conger, J.D. (1993) J. Clin. Invest. 91, 2144.
16. Hyslop, S. and de Nucci, G. (1992) Pharmacol. Res. 26, 223.
17. Wendel, M. et al. (2006) J. Histochem. Cytochem. 54, 1193.
18. Zeng, C. et al. (2008) Kidney Int. 74, 750.
19. Bringmann, A. and Reichenbach, A. (2001) Front. Biosci. 6, 72.
20. Guidry, C. (2005) Prog. Retin. Eyes Res. 24, 75.
21. Rattner, A. and Nathans, J. (2005) J. Neuroscience 25, 4540.
22. Torbidoni, V. et al. (2006) Exp. Biol. Med. 231, 1095.
23. Li, L. et al. (2008) J. Cereb. Blood Flow Metab. 28, 468.
A) and B) No difference is detected in the distribution of astrocytes in the penumbra in the two groups
although astrocytes of GFAP -/-Vim -/- mice show less prominent hypertrophy of cellular processes. Green,
S100β; blue, cell nuclei visualized by ToPro-3. Using Anti-ET-B (#AER-002), reactive astrocytes adjacent to
the ischemic lesion in WT mice (visualized by antibodies against GFAP, C and I) were ET-B positive (E and
K) 7 days later after MCA transection. F) and L) In contrast, no ET-B was detected in astrocytes in GFAP -/-
Vim -/- mice. G) and M) Merged images of (C and E) and of (I and K) respectively. C)-N) Red, GFAP; green,
Related Products
Compound
Cat. #
ET-B; blue, nuclei visualized by ToPro-3. Scale bar, 50 μm.
Antibodies to G-Protein Coupled Receptors
Adapted from reference 23 with permission of Nature Publishing Group.
Anti-ET-A_ _____________________________________ AER-001
Anti-ET-B______________________________________ AER-002
Figure 8. ET-B and Bundles of Intermediate Filaments Colocalize in Cultured WT Astrocytes.
Laser-scanning confocal microscopy
reveals a filamentous appearance of
ET-B immunostaining using Alomone
Labs’ Anti-ET-B (#AER-002), which
colocalizes with GFAP-positive bundles
of intermediate filaments. Red, GFAP;
green, ET-B. Scale bar, 20 μm.
Adapted from reference 23 with
permission of Nature Publishing Group.
6
GPCR Pathways No.3 Winter 2010 www.alomone.com
Muscarinic Receptor Family
Dovrat Brass, Ph.D., B.Pharm.
The muscarinic receptors belong to the G-protein coupled receptors (GPCR) family. In
mammals, at least five different muscarinic acetylcholine receptor subtypes (mAChRs;
m1 through m5) are known to be widely expressed and distributed in different tissues
from different species. They mediate distinct physiological functions according to
their location and receptor subtype. Here, we summarize selected published uses of
Alomone Labs Anti-muscarinic antibodies, which contribute to the understanding of the
physiological aspects of mAChR and their mechanisms of action.
Acetylcholine (Ach), the major neurotransmitter
in the central and peripheral nervous systems
acts through two kinds of receptors: ionotropic
receptors, which consist of rapidly activated
ion channels and metabotropic receptors. The
metabotropic receptors regulate ion channels
and many physiological processes through their
binding and activation of G proteins. The ACh
metabotropic receptors are also called muscarinic
receptors (mAChRs), and are widely distributed
in the plasma membranes of certain neurons and
other mammalian cells.
Five subtypes of muscarinic receptors have
been determined, m1 through m51. They vary in
their distribution and their affinity to various G
proteins, with some correlation according to the
receptor subtype. As a member of the G-protein
coupled receptors (GPCR) family, they mediate
most of the physiological responses to hormones,
neurotransmitters and environmental stimulants.
Like other G-protein coupled receptors, they
have a seven transmembrane domain with an
extracellular N-terminus.
Figure 1. Colocalization of Slack and Slick with Gαq-Protein Coupled Receptors.
Top 12 panels show colocalization of Slick or
Slack with mAChR1, using Anti-m1 antibody
(#AMR-001) in sections of rat cortex (A, frontal
cortex; B, layer V of frontal cortex) and in
hippocampus (C, CA1 region; D, CA3 region).
Slick and Slack immunoreactivity was localized
The use of specific antibodies directed
specifically to each one of the receptors can assist
in elucidating the physiological importance of
these receptors. Three antibodies are offered
by Alomone Labs: Anti-m1 (#AMR-001), Anti-m2
(#AMR-002) and Anti-m3 antibodies (#AMR-006).
GPCR Pathways No.3 Winter 2010 www.alomone.com
with Cy3 (red), and muscarinic receptor
immunoreactivity was visualized using Alexa
Fluor 488 secondary antibody (green). On the
overlaid images, regions of colocalization
appear orange to yellow. Scale bar, 20 µm.
Adapted from reference 3 with permission of The
Society for Neuroscience.
7
M1 Muscarinic Receptors
Figure 2. Kv2.1 is Specifically Associated with Large Cholinergic Terminals.
M1 muscarinic receptors (m1) mediate slow EPSP
(excitatory postsynaptic potential) at the ganglion
in the postganglionic nerve, and are common
in exocrine glands and in the CNS. They are
predominantly found acting via G proteins of class
Gq. The distribution of m1 in RGS9-2 transfected
CHO cells was monitored using Anti-m1 antibody
(#AMR-001) in immunohistochemical analysis.
The research showed colocalization of both m1
and RGS9-2 in these cells2.
Postsynaptic m2 muscarinic receptor (red), identified by
using the Anti-m2 antibody (#AMR-002) is colocalized
with surface membrane Kv2.1-immunoreactivity (KV2.1-IR)
(green). At low and high power (inset), the two channels
Immunohistochemical studies showed that in the
rat cortex and hippocampus the Slack (Slo2.2)
and Slick (Slo2.1) ion channels, members of the
mammalian Slo K+ channel gene family, colocalize
with m13 (Figure 1).
Immunocytochemical experiments performed in
additional work, indicated that the m1 muscarinic
receptor subtype is present in both rat and
mudpuppy taste cells4.
of fluorescence signal overlap almost completely at the
locations of large clusters of Kv2.1 (yellow, inset).
Adapted from reference 6 with permission of Blackwell
Publishing Ltd.
Figure 3. M2 mAChR and Golgi Zone Staining in HEK293 Cells.
M2 Muscarinic Receptors
M2 muscarinic receptors (m2) are expressed
in the heart, where they act to slow the heart
rate down to normal sinus rhythm by slowing
the speed of depolarization after stimulatory
actions of the parasympathetic nervous system.
They also reduce contractile forces of the atrial
cardiac muscle, and reduce conduction velocity
of the atrioventricular node (AV node). M2
muscarinic receptors act via G proteins of class
Gi. Immunoprecipitation studies using Anti-m2
antibody (#AMR-002) showed that m2 is not
associated with the native rat atrial KACh channels
complex, composed of GIRK1-Gβ complex,
GIRK2/3, PKAc, PP1, PP2A, and RACK1 proteins5.
Additional work using confocal microscopy,
showed that Kv2.1 K+ channel expression is
enriched at synapses formed by cholinergic
C-terminals, and that the Kv2.1 subunit-containing
channels are colocalized with postsynaptic m26
(Figure 2). Anti-m2 antibody was also used in
immunoprecipitation studies to monitor the effect
of receptor stimulation on m2 protein levels in
atrial membrane. It was demonstrated that G
protein-regulated inwardly rectifying K+ (GIRK)
channels can operate as dynamic integrators
of α-Adrenergic and cholinergic signals in rat
atrial myocytes7. It was also demonstrated in
HEK293 cells, stably expressing m2 receptors
(using immunocytochemical studies) that
the internalization of m2 is modulated by the
activation of heterotrimeric G proteins and
is directed mainly to the Golgi8 (Figure 3).
Immunofluoresence showed that expression of
m2 is widespread in sections of E17 cerebral rat
cortex embryos. M2 receptors were found to be
distributed in the ventricular, subventricular and
cortical plate zones, indicating that these cells are
8
M2 mAChR
Golgi
Costaining
HEK293 cells stably expressing m2 mAChRs were fixed and stained with Anti-m2 antibody (#AMR-002) (A) and anti-Golgi
zone antibodies (B) and visualized with TRITC-conjugated anti-rat antibodies and FITC-conjugated anti-mouse antibodies.
C) An overlay of the corresponding m2 mAChR and Golgi zone images (yellow color indicates colocalization).
Adapted from reference 8 with permission of the American Society for Pharmacology & Experimental Therapeutics.
Figure 4. Muscarinic Acetylcholine Receptor Expressed in the Ventricular (vz) and
Subventricular (sv) Zones of the Embryonic Cortex.
A
B
Nissl staining (A) and immunofluorescence using
Anti-m2 antibody (#AMR-002) (B) show a widespread
distribution of m2 AChR-immunoreactive cells in the
vz and sv zones and cortical plate (CP) of coronal
sections of E17 rat cortex. Scale bar, 150 µm.
Adapted from reference 9 with permission of The
Society for Neuroscience.
GPCR Pathways No.3 Winter 2010 www.alomone.com
proliferating neuroepithelial cells9 (Figure 4).
class Gq. Anti-m3 antibody (#AMR-006) was used,
in immunocytochemistry studies, to confirm
In resting cardiomyocytes, m2 and β1-AR
Adrenergic Receptors have a similar membrane
distribution, which was demonstrated by western
blot analysis. In contrast, β2-ARs and m2 largely
segregate to separate membrane subdomains10.
that mesencephalic GABAergic neurons can be
Using Anti-m2 antibody in immunoprecipitation
studies, the collision-coupling mechanism
between m2 and G proteins was assessed.
Results of this study suggest a stoichiometric
coupling between the G protein and GIRK as the
effectors in Xenopus oocytes11.
immunocytochemistry analysis at the plasma
Additional work proposed, by western blot
analysis, that the acidic amino acids at sites A1
and A2 in m2 play important roles in agonistdependent phosphorylation at sites P2 and P1,
respectively, and also play an important role in
interactions of arrestin with m212.
M4 Muscarinic Receptors
regulated directly through muscarinic receptors13
Figure 5. The Effects of Muscarine Involve
M3 Muscarinic Receptors in Mesencephalic
GABAergic Neurons.
(Figure 5).
Additionally, m3 is detected using
membrane of human ASM (airway smooth muscle)
cells containing caveolin-1. Caveolin-1 contributes
to regulation of [Ca2+]i responses to agonists in
these cells14 (Figure 6).
M4 muscarinic receptors (m4) are found mainly
in the CNS. These receptors act via G proteins of
Image showing the result of a double-labelling
class Gi.
immunofluorescence experiment performed using Anti-m3
antibody (#AMR-006) (red) and a tyrosine hydroxylase (TH)
antibody (green). Scale bar, 15 µm. M3-like immunoreactivity
M3 Muscarinic Receptors
The M5 Muscarinic Receptors
M3 muscarinic receptors (m3) are located in the
smooth muscles of the blood vessels, as well as
in the lungs. M3 receptors act via G proteins of
The M5 muscarinic receptors (m5) distribution is
is observed in non-TH (presumed GABAergic) neurons.
Adapted from reference 13 with permission of Blackwell
Publishing Ltd.
not well known. Like the m1 and m3 receptors, m5
receptors are coupled with G proteins of class Gq.
References
Figure 6. M3 Receptor and Caveolin-1 (Cav-1) Colocalize in Human Airway Smooth
Muscle (ASM) Cells.
1. Wess, J. (1993) Life Sci. 53, 1447.
2. Kovoor, A. et al. (2005) J. Neurosci. 25, 2157.
3. Santi, C.M. et al. (2006) J. Neurosci. 26, 5059.
4. Ogura, T. (2002) J. Neurophysiol. 87, 2643.
5. Nikolov, E.N. and Ivanova-Nikolova, T.T. (2004) J. Biol. Chem. 279,
23630.
6. Muennich, E.A.L. and Fyffe, R.E.W. (2003) J. Physiol. 554, 673.
7. Nikolov, E.N. and Ivanova-Nikolova, T.T. (2007) J. Biol. Chem. 282,
28669.
8. Roseberry, A.G. et al. (2001) Mol. Pharmacol. 59, 1256.
9. Li, B.S. et al. (2001) J. Neurosci. 21, 1569.
10. Rybin, V.O. et al. (2000) J. Biol. Chem. 275, 41447.
11. Vorobiov, D. et al. (2000) J. Biol. Chem. 275, 4166.
12. Lee, K.B. et al. (2000) J. Biol. Chem. 275, 35767.
13. Michel, F.J. et al. (2004) J. Physiol. 556, 429.
14. Prakash, Y.S. et al. (2007) Am. J. Physiol. 293, L1118.
Related Products
Two-color immunocytochemistry and laser scanning confocal microscopy were used to localize caveolin-1
Compound
(green, Cy3) predominantly to the plasma membrane of ASM cells. Caveolin-1 colocalized with several
Antibodies
intracellular Ca2+ concentration ([Ca2+]i)-regulatory proteins (red, Cy5), including receptors for Ach (m3 receptor)
using Anti-m3 antibody (#AMR-006) (shown at X1,000 magnification). Scale bar, 1 µm, except in top row (10 µm).
Adapted from reference 14 with permission of the American Physiological Society.
GPCR Pathways No.3 Winter 2010 www.alomone.com
Cat. #
Anti-m1_ ______________________________________ AMR-001
Anti-m2 _______________________________________ AMR-002
Anti-m3_ ______________________________________ AMR-006
9
Exploring P2Y Receptors
Ofra Gohar, Ph.D.
Following Hypoxia or inflammation, extracellular nucleotide release can activate two
families of receptors, the ionotropic P2X receptors with seven subtypes (P2X1-7), and
the metabotropic G-protein coupled P2Y receptors, which are the focus of this review.
Eight mammalian P2Y receptors have been identified on the basis of sequence homology
and pharmacological profile: the P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and
P2Y14. Alomone Labs has been and remains quite active in this field of research and this
brief report summarizes how Alomone Labs’ P2Y products have influenced the advances
made.
Figure 1. Expression of P2Y1 Receptor in Human Colonic Circular Smooth Muscle.
P2Y Receptors
Hypoxia or inflammation, activates the release
of extracellular nucleotides such as ATP and UTP
which interact with cell surface P2Y receptors to
produce a broad range of physiological responses
including cardiac function, platelet aggregation
and smooth muscle cell proliferation1, 2.
Two subfamilies are defined within the P2Y family,
which are distinct from each other on the basis of
sequence identity and signaling properties:
The P2Y1 subfamily includes the P2Y1, P2Y2,
P2Y4, P2Y6 and P2Y11 receptors and is coupled to
phospholipase C, mediating increases in inositol
triphosphate (IP3).
The P2Y12 subfamily consists of the P2Y12,
P2Y13, and P2Y14 receptors and is coupled to the
inhibition of adenylyl cyclase.
P2Y receptors are widely distributed in the
nervous system, as well as in many other
peripheral tissues. All P2Y receptor subtypes
are capable of modulating ion channels (with
the exception of P2Y14 that has not been
characterized yet)1.
Subtype specific antibodies provide a very
powerful tool for exploring tissue distribution,
expression and localization of the receptors by
different methods such as Western Blotting,
Immunohistochemistry, Immunocytochemistry
and Flow Cytometry. Alomone Labs' P2Y
antibodies provide a powerful tool in exploring
the various roles of these receptors.
10
Immunohistochemical staining of P2Y1 in colonic circular smooth muscle (positive and control) in the
absence of the primary antibody (A) colonic longitudinal smooth muscle (positive sample and control)
(B) and myenteric ganglia (positive sample and control) (C) using Anti-P2Y1 antibody (#APR-009).
Adapted from reference 12 with permission of the American Physiological Society.
GPCR Pathways No.3 Winter 2010 www.alomone.com
The P2Y1 Subfamily: P2Y1, P2Y2,
P2Y4, P2Y6 and P2Y11 Receptors
Figure 2. Expression of P2Y1 Receptor in Rat Duodenum.
All receptors of this subfamily have been
cloned and characterized. Pharmacological
characterization of P2Y1 showed that in contrast
to the other receptors, ADP is a more potent
agonist than ATP3.
In platelets, P2Y1 is co-expressed with P2Y12,
and is responsible for the initial shape change,
aggregation, and rise in intracellular Ca2+ upon
activation in response to ADP4.
P2Y1 was isolated from different species
including rat, mouse, bovine, and human and its
expression was found to be widely distributed
along with that of P2Y2 and P2Y4 in Dorsal Root
Ganglion (DRG)5,6, bladder7, duodenum8, retina9-11,
colon12 (Figure 1) and various cell lines8,13, using
Anti-P2Y1 (#APR-009) antibody.
Western blot analysis and immunohistochemistry
studies (for the subepithelial fibroblast) show
that rat duodenum8 (Figure 2), primary culture
of subepithelial fibroblast, 18Co cells and rat
cerebrum, express P2Y1. In primary culture of
rat DRGs, co-localization of P2Y1 and other ion
channels was shown using Anti-P2Y1 antibody
in immunocytochemistry5. This study shows that
activation of P2Y1 inhibits voltage-activated
Ca2+ channels in DRGs and might be involved in a
mechanism of ADP-induced analgesia5. Activation
of P2Y1 also induces the inhibition of the
M-Type K+ currents in rat hippocampal pyramidal
neurons14. Overexpression of P2Y1 in HEK293
cells was assessed by immunocytochemistry
using Anti-P2Y1 antibody14 (Figure 3).
Immunohistochemical and immunocytochemical staining of P2Y1 in rat duodenum and primary culture of subepithelial
fibroblasts using Anti-P2Y1 antibody (#APR-009). Cryosections of 6W Wistar rat duodenum were incubated with A)
Anti-P2Y1 antibody and B) Anti-P2Y1 antibody pre-absorbed with 2 μg ml-1 antigenic peptide, followed with anti-rabbit
Envision+ and visualized with DAB-H2O2 reactions. (C) Subepithelial fibroblasts isolated from rat duodenal villi in
primary cultures were incubated with Anti-P2Y1 antibody, then biotinylated anti-rabbit IgG, followed by streptavidinTexas red. Scale bar in A and B is 100 μm, and in C is 10 μm.
Adapted from reference 8 with permission of The Company of Biologists.
Figure 3. Expression of P2Y1 Receptor in HEK293 Cells Stably Expressing Myc-Tagged
P2Y1 Receptors.
Immunocytochemical staining of P2Y1 in HEK293 cells stably expressing Myc-tagged P2Y1 receptors. Cells were
co-reacted with anti-Myc antibody (red) and with Anti-P2Y1 antibody (#APR-009). Reaction was completely
blocked by its antigen peptide.
Adapted from reference 14 with permission of The Society for Neuroscience.
Figure 4. Expression of P2Y2 and P2Y4 Receptors in Cat Dorsal Root Ganglia.
P2Y2 receptor is abundantly expressed in
the apical membranes of several epithelial
tissues including airway5. It was reported to be
expressed, using Anti-P2Y2 antibody (#APR-010),
in mouse mesangial cells15, human intestinal
epithelium carcinoma cells13, DRGs6 (Figure 4),
hippocampus16, retina9,11 (Figures 5 and 6) and
bladder7,17, 18 (Figure 7).
The involvement of P2Y2 in enhanced neuronal
differentiation was demonstrated in mouse
DRGs and in PC12 cells. Activation of P2Y2
receptor by ATPγS enhanced NGF-dependent
neuronal differentiation and neurite extension19.
Expression levels of P2Y2 in rat DRG P2Y2 -/- cells
or in PC12 cells treated with siRNA targeted
against P2Y2, were assessed by western blot
analysis using Anti-P2Y2 antibody19 (Figures 8
and 9).
The P2Y4 receptor is highly expressed in the
placenta and to lower extent in lungs and vascular
smooth muscles20.
Immunohistochemical staining of P2Y2 and P2Y4 in cat Dorsal Root Ganglia (DRG) using Anti-P2Y2
Human P2Y4 receptor is highly selective to UTP,
Adapted from reference 6 with permission of the Histochemical Society.
GPCR Pathways No.3 Winter 2010 www.alomone.com
(#APR-010) and Anti-P2Y4 (#APR-006) antibodies (red) with IB4, CGRP or NF200 (green).
11
while rat P2Y4 receptor is equally activated by
both UTP and ATP and also by other triphosphate
nucleotides20.
Figure 5. Expression of P2Y Receptors in Human Retina.
Expression of P2Y4 and P2Y6 receptors in the
intramural ganglia of cat urinary bladder7 (figure
7) and human retina9 (figures 5 and 6) was
demonstrated using Anti-P2Y4 (#APR-006) and
Anti-P2Y6 (#APR-011) antibodies respectively.
The regulation of ion channels by P2Y receptors
was described in several reports. Activation of
P2Y receptors by UTP in immortalized human
mammary epithelial cells enhanced Na+ transport
by activating KCa3.1 (KCNN4) channels21. In this
study, the expression of P2Y receptors was
confirmed by western blot analysis using the
following Anti-P2Y receptors antibodies: AntiP2Y1, Anti-P2Y2, Anti-P2Y4, Anti-P2Y6, and AntiP2Y11 (#APR-015) antibodies21.
The P2Y11 receptor is the only subtype coupled
to the activation of adenylyl cyclase and PLC,
leading to the accumulation of cAMP and inositol
phosphates22.
The P2Y12 Subfamily: P2Y12,
P2Y13, and P2Y14 Receptors
Expression of P2Y12 and P2Y13 in cholangiocytes
(the epithelial cells lining intrahepatic bile
ducts) was demonstrated, for the first time, by
using Anti-P2Y12 (#APR-012) and Anti-P2Y13
(#APR-017) antibodies in western blots and in
immunohistochemical staining (confocal and
electron microscopy)23 (Figure 10). Apically
applied nucleotides affected the forskolininduced cAMP increase in rat cholangiocytes
in a P2Y12-dependent manner. Incubation of
the epithelial cells lining intrahepatic bile ducts
with siRNA to P2Y12 abolished the effects of
ADP on forskolin-induced cAMP levels, as well
as P2Y12 protein levels which were confirmed
by western blot analysis23. Expression of P2Y12
was also demonstrated in different tissues;
western blot analysis showed that in rat spinal
cord, P2Y12 expression is upregulated following
partial sciatic nerve ligation (using Anti-P2Y12
antibody) and by immunohistochemical
staining24. P2Y12 expression was also shown
in intramural ganglia of the cat urinary bladder
by immunohistochemistry18, in vascular smooth
muscle cells and platelets by western blot, where
it was also shown that P2Y12 receptor stimulation
mediates contraction in human blood vessels25.
Retinal slices were stained against P2Y1 (A1), P2Y2 (B1), P2Y4 (C1) and P2Y6 (D1) receptors using Anti-P2Y1
(#APR-009), Anti-P2Y2 (#APR-010), Anti-P2Y4 (#APR-006) and Anti-P2Y6 (#APR-011) antibodies (red). Vimentin,
a retinal marker (green) was detected (A2, B2, C2, D2). Areas that express both, receptor and vimentin, are shown
in yellow (A3, B3, C3, D3). Cell nuclei were stained with Hoechst 33258 (blue). NFL, nerve fiber layer; INL, inner
nuclear layer; ONL, outer nuclear layer.
Adapted from reference 9 with permission of the Association for Research in Vision and Ophthalmology.
Figure 6. Expression of P2Y Receptors in Porcine Retina.
Immunohistochemical staining of P2Y receptors in the porcine retina using Anti-P2Y1 (#APR-009), Anti-P2Y2 (#APR010), Anti-P2Y4 (#APR-006) antibodies. Slices were derived from nonsurgical control retinas, vitrectomized control
P2Y14 is expressed in several tissues including
brain, lung and several cells of hematopoietic
origin such as T lymphocytes and dendritic cells.
The exact function of this receptor in the different
tissues has not been clearly established but
an involvement in chemotaxis, maturation of
immature dendritic cells and neuroimmune
regulation has been suggested1.
12
retinas and from detached and attached areas of porcine retinas at 7 days after surgery.
Adapted from reference 11 with permission of the Association for Research in Vision and Ophthalmology.
GPCR Pathways No.3 Winter 2010 www.alomone.com
Figure 7. Expression of P2Y Receptors in Cat Urothelium.
Expression of P2Y12 in Mouse Brain.
A
B
Immunohistochemical staining of P2Y12 (red) in mouse brain
using Anti-P2Y12 antibody (#APR-012). Counterstaining with
sytox green (green). The P2Y12 positive fibers (arrows) run
from the corpus callosum (triangles) into the deep layers of
the entorhinal cortex.
Western blot analysis of rat brain membranes (1,2)
or human platelets (3,4):
1,3. Anti-P2Y12 antibody (#APR-012), (1:200).
2,4. Anti-P2Y12 antibody, preincubated with the
control peptide antigen.
A) Localization of P2Y2 using Anti-P2Y2 antibody (#APR-010) in normal cat uorothelium (left),
CYTK-20, a marker for urothelial cells (middle) and representative merged image illustrates
the colocalization of P2Y2 and CYTK-20 as well as DAPI (nuclear marker). B) Localization of
P2Y1, P2Y2 and P2Y4 in normal (left) and FIC (right) cat urothelium using Anti-P2Y1 (#APR009), Anti-P2Y2 and Anti-P2Y4 (#APR-006) antibodies.
Adapted from reference 7 with permission of the American Physiological Society.
Figure 8. Expression of P2Y2 Receptor in Mouse DRG Neurons.
Figure 9. Expression of P2Y2 Receptor in Rat PC12 Cells.
Western blot analysis of P2Y2 expression in PC12 cells treated with P2Y2
Western blot analysis of P2Y2 expression in DRG neurons from wild-
siRNA or scrambled sequence. P2Y2 expression and down-regulation was
type and P2Y2 -/- P1 mice using Anti-P2Y2 antibody (#APR-010).
assessed with Anti-P2Y2 antibody (#APR-010).
Adapted from reference 19 with permission of The National Academy of
Adapted from reference 19 with permission of The National Academy of
Sciences of the USA (copyright 2005).
Sciences of the USA (copyright 2005).
GPCR Pathways No.3 Winter 2010 www.alomone.com
13
Figure 10. Expression of P2Y12 and P2Y13 in Rat Cholangiocytes.
Related Products
Compound
A
Cat. #
Antibodies to P2Y G-Protein Coupled Receptors
Anti-P2Y1______________________________________
Anti-P2Y2______________________________________
Anti-P2Y4______________________________________
Anti-P2Y6______________________________________
Anti-P2Y11____________________________________
Anti-P2Y12_ ___________________________________
Anti-P2Y13_ ___________________________________
Anti-P2Y14 (extracellular)_ ______________________
B
APR-009
APR-010
APR-006
APR-011
APR-015
APR-012
APR-017
APR-018
Antibodies to P2X Purinergic Receptors
Anti-P2X1_ ____________________________________
Anti-P2X2_ ____________________________________
Anti-P2X3_ ____________________________________
Anti-P2X4_ ____________________________________
Anti-P2X5_ ____________________________________
Anti-P2X6_ ____________________________________
Anti-P2X7_ ____________________________________
Anti-P2X7-ATTO-550____________________________
Anti-P2X7 (extracellular)_ _______________________
Anti-P2X7 (extracellular)-FITC____________________
C
APR-001
APR-003
APR-016
APR-002
APR-005
APR-013
APR-004
APR-004-AO
APR-008
APR-008-F
PKC Activator
PMA_ _________________________________________ P-800
Adenylate Cyclase Activator
Forskolin______________________________________ F-500
A) Western blot analysis of P2Y12 and P2Y13 expression in cholangiocytes, hepatocytes and brain using AntiP2Y12 (#APR-012) and P2Y13 (#APR-017) antibodies. B) Immunohistochemical staining in rat liver and immunogold
scanning electron microscopy (C) of P2Y12 cholangiocytes cilia.
Adapted from reference 23 with permission of the American Physiological Society.
References
1. Abbracchio, M.P. et al. (2006) Pharmacol. Rev. 58, 281.
2. Wang, L. et al. (2002) J. Cardiovas. Pharmacol. 40, 841.
3. Vassort, G. (2001) Physiol. Rev. 81, 767.
4. Jin, J. et al. (1998) J. Biol. Chem. 273, 2030.
5. Gerevich. Z. et al. (2004) J. Neurosci. 24, 797.
6. Ruan, H.Z. et al. (2005) J. Histochem. Cytochem. 53, 1273.
7. Birder, L.A. et al. (2004) Am. J. Physiol. 287, F1084.
8. Furuya, K. et al. (2005) J. Cell Sci. 118, 3289.
9. Fries, J.E. et al. (2005) Invest. Ophthalmol. Vis. Sci. 46, 3000.
Activation of Adenylate Cyclase (in vivo) by Forskolin in NIH/C6 Neuroblastoma Cells.
10. Fries, J.E. et al. (2004) Invest. Ophthalmol. Vis. Sci. 45, 3410.
11. Iandiev, I. et al. (2006) Invest. Ophthalmol. Vis. Sci. 47, 2161.
12. Gallego, D. et al. (2006) Am. J. Physiol. 291, G584.
13. Coutinho-Silva, R. et al. (2005) Am. J. Physiol. 288, G1024.
14. Filippov, A.K. et al. (2006) J. Neurosci. 26, 9340.
15. Rivera, I. et al. (2007) Am. J. Physiol. 292, F1380
16. Rodrigues, R.J. et al. (2005) J. Neurosci. 25, 6286.
17. Chopra, B. et al. (2008) Am. J. Physiol. 294, F821.
18. Ruan, H.Z. et al. (2006) Am. J. Physiol. 290, F1143.
19. Arthur, D.B. et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 19138.
20. von Kugelgen,I. and Wetter, A. (2000) Naunyn-Schmiedebergs
Arch Pharmacol. 362, 310
NIH/C6 cells were pre-incubated for 30 min
with IBMX (10 µM), and then stimulated for
30 min with Forskolin (#F-500) (0-100 µM).
21. Tovell, V,E. and Sanderson, J. (2008) Invest. Ophthalmol. Vis. Sci.
49, 350.
22. Communi, D. et al. (1997) J. Biol. Chem. 272, 31969.
The cell's content of cAMP was measured
23. Masyuk, A.I. et al. (2008) Am. J. Physiol. 295, G725.
by an ELISA kit (Amersham).
24. Kobayashi, K. et al. (2008) J. Neurosci. 28, 2892.
25. Wihlborg, A-K. et al. (2004) Arterioscler. Thromb. Vasc. Biol. 24,
1810
14
GPCR Pathways No.3 Winter 2010 www.alomone.com
GPCR Product List
5-hydroxytryptamine Receptors (Serotonin
Receptors)
Anti-D3 Dopamine Receptor (ext.)
ADR-003
Anti-Neurotensin Receptor 2
ADR-005
Opioid Receptors
Anti-5-Hydroxytryptamine
Receptor 1A
Anti-D5 Dopamine Receptor (ext.)
Endothelin Receptors
ASR-021
Adenosine Receptor
Anti-A1 Adenosine Receptor
AAR-006
Anti-A2A Adenosine Receptor
AAR-002
Anti-A2B Adenosine Receptor (ext.) AAR-003
Anti-A3 Adenosine Receptor
AAR-004
Anti-α1A-Adrenoceptor (ext.)
AAR-015
Anti-α1B-Adrenoceptor (ext.)
AAR-018
Anti-α1B-Adrenoceptor (ext.)ATTO-488
AAR-018-AG
Anti-β2-Adrenoceptor (ext.)
Anti-β3-Adrenoceptor (ext.)
Anti-Angiotensin-(1-7) Mas
Receptor
Orexin Receptors
Anti-Orexin Receptor 2
AGB-002
Prokineticin Receptors
Anti-Galanin Receptor Type 1
AGR-011
APR-031
Anti-Proteinase-activated
Receptor-2
APR-032
Anti-Proteinase-activated
Receptor-4 (ext.)
APR-034
Anti-Proteinase-activated
Receptor-4 (ext.)-FITC
APR-034-F
AGC-007
Histamine Receptors
Anti-H1 Histamine Receptor
AHR-001
Anti-Rat H2 Histamine Receptor
(ext.)
AHR-002
Anti-H3 Histamine Receptor
AHR-003
AAR-011-AO
Anti-Human H4 Histamine
Receptor (ext.)
AHR-004
AAR-012
Kisspeptins Receptor
Anti-Bombesin Receptor 1
ABR-004
Anti-Bombesin Receptor 2 (ext.)
ABR-002
Anti-Bombesin Receptor 3 (ext.)
ABR-003
Bradykinin Receptors
Anti-Kisspeptins Receptor (ext.)
AKR-001
Melanocortin Receptors
Anti-Melanocortin Receptor 1
AMR-020
Anti-Melanocortin Receptor 2
(ext.)
AMR-022
Anti-Melanocortin Receptor 3
(ext.)
AMR-023
ABR-011
Anti-Melanocortin Receptor 4
(ext.)
AMR-024
Anti-B2 Bradykinin Receptor
ABR-012
Anti-Melanocortin Receptor 5
AMR-025
Anti-Calcium Sensing Receptor
(ext.)
Muscarinic Receptors
ACR-004
Cannabinoid Receptors
Anti-Cannabinoid Receptor 1 (ext.) ACR-001
Anti-Cannabinoid Receptor 2
ACR-002
Anti-Cannabinoid Receptor 2 (ext.) ACR-003
Cholecystokinin Receptors
Anti-Cholecystokinin B Receptor
(ext.)
ACR-042
Anti-D2 Dopamine Receptor (ext.)
Anti-m1
AMR-001
Anti-m2
AMR-002
Anti-m3
AMR-006
Neuropeptide Y Receptors
ADR-002
GPCR Pathways No.3 Winter 2010 www.alomone.com
Anti-P2Y1
APR-009
Anti-P2Y2
APR-010
Anti-P2Y4
APR-006
Anti-P2Y6
APR-011
Anti-P2Y11
APR-015
Anti-P2Y12
APR-012
Anti-P2Y13
APR-017
Anti-P2Y14 (ext.)
APR-018
Somatostatin Receptors
ASR-001
Anti-Somatostatin Receptor Type 2
ASR-006
(ext.)
Anti-Somatostatin Receptor Type 3 ASR-003
Anti-Somatostatin Receptor Type 4
ASR-004
(ext.)
Anti-Somatostatin Receptor Type 5
ASR-005
(ext.)
Sphingosine-1-Phosphate Receptors
Anti-Sphingosine 1-Phosphate
Receptor 1 (ext.)
Anti-Neuropeptide Y1 Receptor
ANR-021
Urotensin II Receptor
Anti-Neuropeptide Y2 Receptor
ANR-022
Anti-Urotensin II Receptor
Anti-Neuropeptide Y4 Receptor
ANR-024
Anti-Neuropeptide Y5 Receptor
ANR-025
Neurotensin Receptors
Dopamine Receptors
Purinergic (P2Y) Receptors
Anti-Somatostatin Receptor Type I
Anti-B1 Bradykinin Receptor
Calcium Sensing Receptor
APR-042
Anti-Human Proteinase-activated
Receptor-1 (ext.)
Anti-mGluR5 (ext.)
Bombesin Receptors
Anti-Prokineticin Receptor-2 (ext.)
Glutamate Receptors
AGC-012
AAR-012-AG
APR-041
Proteinase-activated Receptors
Anti-mGluR3 (ext.)
Anti-Angiotensin II Receptor
Type‑2 (ext.)
Anti-Prokineticin Receptor-1 (ext.)
Anti-Galanin Receptor Type 3 (ext.) AGR-013
AAR-017
Anti-Angiotensin II Receptor
Type‑1 (ext.)-ATTO-550
AOR-002
AGB-001
AGC-011
AAR-011
AOR-001
Anti-GABA (B) R2
Anti-mGluR2 (ext.)
Anti-Angiotensin II Receptor
Type‑1 (ext.)
Anti-Orexin Receptor 1
Anti-GABA (B) R1 (ext.)
AAR-016
Angiotensin II Receptors
Anti-Angiotensin II Receptor
Type‑2 (ext.)-ATTO-488
Anti-µ-Opioid Receptor (ext.)
AER-002
AGC-006
AAR-013
AOR-011
AER-001
Anti-mGluR1 (ext.)
Angiotensin-(1-7) Mas Receptor
AOR-014
Anti-ET-B
Galanin Receptors
Adrenoceptors
Anti-δ-Opioid Receptor (ext.)
Anti-ET-A
GABA (B) Receptors
ANT-016
ASR-011
AER-003
Primary antibody conjugated to a fluorescent
dye under license from ATTO-TEC GmbH.
ext. = extracellular
Anti-Neurotensin Receptor 1 (ext.) ANT-015
15
Stranded? Let us help.
Quality Cell
Signaling Tools at Alomone Labs
Adenylate Cyclase Activator
Forskolin............................................................. F-500
Protein Kinase Inhibitors (non specific)
SOS
Staurosporine......................................................S-350
PKA Inhibitor
KT5720................................................................K-190
PKC Activator
PMA.P-800
PKC Inhibitor
Chelerythrine..................................................... C-400
MAPK and MAPK Homolog Activator
Anisomycin........................................................ A-520
MAPK and MAPK Homolog Inhibitors
PD 98059........................................................... P-260
SB 203580...........................................................S-370
U0126.................................................................U-400
PI3-Kinase Inhibitors
LY 294002........................................................... L-300
Wortmannin...................................................... W-400
CDK Inhibitors
Olomoucine........................................................O-300
Roscovitine......................................................... R-300
Protein Phosphatase Inhibitors (PP-1 and PP-2A)
Calyculin A..........................................................C-100
Okadaic Acid (ammonium salt)........................O-800
Okadaic Acid (sodium salt)...............................O-900
Histone Deacetylase Inhibitor
Trichostatin A...................................................... T-300
Mitochondrial ATPase Inhibitor
Oligomycin.........................................................O-500
Ca2+ Ionophores
A23187 .............................................................A-600
Ionomycin........................................................... I-700
Intracellular Ca2+ Mobilizers
Cyclopiazonic Acid..............................................C-750
Imperatoxin A..................................................... I-300
rMaurocalcine................................................RTM-100
Ryanodine.......................................................... R-500
Thapsigargin.......................................................T-650
Molecular Tools for the Life Science Community
A complete list of cell signaling products could be
found at www.alomone.com.
ATTO Technology
For Direct Immunohistochemical & Immunocytochemical Applications
Alomone Labs is pleased to offer a new line of its well-characterized antibodies directly conjugated to a new generation of specially
developed, bright fluorescent dyes by ATTO-TEC.
ATTO dyes are well known in the field of fluorescent technology and are characterized by strong absorption (high extinction
coefficient), high fluorescence quantum yield, and high photo-stability. ATTO dyes are analogous to the established Alexa dyes and
were found to be comparable to any fluorescent technology in the market.
The proven quality of the Alomone labs' antibodies together with the bright ATTO dyes generate an invaluable tool that undergoes
careful quality control and is specially suited for applications that require simultaneous labeling of different markers.
Alomone Labs is currently offering primary antibodies directly conjugated to the following ATTO dyes: ATTO-488 (green) and
ATTO-550 (orange).
Label
Fluorescence
Spectra
Alternative to
ATTO-488
Green
λex 498 nm; λem 520 nm in 0.1 M phosphate pH 7.0
FITC, Alexa-488
ATTO-550
Orange
λex 554 nm; λem 576 nm in 0.1 M phosphate pH 7.0
TAMRA, Cy3, Alexa-555
The ATTO dyes are compatible with common fluorescence instrumentation.
ATTO-488 is a novel fluorescent label with excellent
water solubility. Characteristic features of the label are
strong absorption, high fluorescence quantum yield,
high photo-stability, and very little triplet formation.
The fluorescence is excited most efficiently in the
range 480 - 515 nm. For instance the 488 nm line of
the Argon-Ion laser is very suitable for excitation of
the ATTO-488 dye.
GPCR Pathways No.3 Winter 2010 www.alomone.com
ATTO-550 is a novel fluorescent label related to the
well-known dyes Rhodamine 6G and Rhodamine
B. Characteristic features of the label are strong
absorption, high fluorescence quantum yield,
and high thermal and photo-stability. The dye is
moderately hydrophilic. ATTO-550 is a cationic dye.
The fluorescence is excited most efficiently in the
range 540 - 565 nm.
17
Fluorescently Labeled GPCR Antibodies
For Direct Immunohistochemical & Immunocytochemical Applications
Anti-Angiotensin II Receptor Type-2
Anti-α1B-Adrenoceptor-ATTO-488
(extracellular)-ATTO-488
(extracellular)
Cat.#: AAR-012-AG
Cat. #: AAR-018-AG
Expression of Angiotensin II Receptor Type-2 in
mouse 3T3-L1 cells
Immunocytochemical staining of α1B-Adrenoceptor
in living GH3 cells
Immunocytochemical staining of intact live mouse 3T3-L1
Immunocytochemical staining of GH3 cells with Anti-α1B-
cells with Anti-Angiotensin II Receptor Type-2 (extracellular)-
Adrenoceptor-ATTO-488 (extracellular) antibody (#AAR-018-
ATTO-488 antibody (#AAR-012-AG), (green), (1:50). Live view
AG), (1:100), (green). Nuclear staining of cells using the cell-
of the same field was superimposed to the fluorescent one.
permeable dye Hoechst 33342 (blue).
Anti-Angiotensin II Receptor Type-1
(extracellular)-ATTO-550
Cat.#: AAR-011-AO
Expression of Angiotensin II Receptor Type-1 in rat C6 glioma cells
A
B
Immunocytochemical staining of live intact rat C6 glioma cells with Anti-Angiotensin II Receptor Type-1 (extracellular)-ATTO-550 antibody (#AAR-011-AO) (A). Live view
of the same field (B).
18
GPCR Pathways No.3 Winter 2010 www.alomone.com
Fluorescently Labeled GPCR Antibodies
For Direct Flow Cytometry Analysis
Flow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric
analysis of the physical and/or chemical characteristics of single cells flowing through an optical and/or electronic detection apparatus. The flow cytometer
was developed in the 1970’s and rapidly became an essential instrument for biological studies. Extracellular antibodies directly labeled with fluorescein are an
important tool for this application.
Anti-Proteinase-activated Receptor-4
Anti-α1B-Adrenoceptor-ATTO-488
(extracellular)-FITC
(extracellular)
Cat. #: APR-034-F
Cat. #: AAR-018-AG
Flow cytometry analysis of Proteinase-activated
Receptor-4 in live intact HL-60 cells
Flow cytometry analysis of α1B-Adrenoceptor in
intact living GH3 cells
Unstained GH3 cells.
GH3 cells + Anti-α1B-Adrenoceptor-ATTO-488
(extracellular) antibody (#AAR-018-AG) (10µg/5x105 cells).
Unstained HL-60 cells.
HL-60 cells + Anti-Proteinase-activated Receptor-4
(extracellular)-FITC antibody (#APR-034-F).
GPCR Pathways No.3 Winter 2010 www.alomone.com
19
NEW GPCR PRODUCTS
Name
Cat. #
5-Hydroxytryptamine Receptor
Anti-5-Hydroxytryptamine Receptor 1A
ASR-021
Name
Cat. #
Anti-A1 Adenosine Receptor
Cat. #: AAR-006
Adenosine Receptors
Anti-A1 Adenosine Receptor
AAR-006
Sizes: 50 µl | 0.2 ml
Anti-A2A Adenosine Receptor
AAR-002
Host: Rabbit.
Anti-A2B Adenosine Receptor
(extracellular)
AAR-003
Anti-A3 Adenosine Receptor
AAR-004
Epitope: Peptide (C)KKVSASSG DPQKYYGKE,
corresponding to amino acid residues 213-229 of
human A1 Adenosine Receptor (Accession P30542).
Applications: WB,IH, IC
Anti-5-Hydroxytryptamine Receptor 1A
Cat. #: ASR-021
Sizes: 50 µl | 0.2 ml
Anti-A2A Adenosine Receptor
Expression of A1 Adenosine Receptor in rat cortex
Cat. #: AAR-002
A
B
Host: Rabbit.
Epitope: Peptide KKSLNGQPGSGDWRRC,
corresponding to amino acid residues 251-266 of rat
5-Hydroxytryptamine Receptor 1A (Accession P19327).
Applications: WB, IH
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide (C)RQLKQMESQPLPGER,
corresponding to amino acid residues 201-215 of
mouse A2A Adenosine Receptor (Accession Q60613).
C
Applications: WB, IH
Expression of 5-Hydroxytryptamine Receptor
1A in rat cerebellum
Expression of A2A Adenosine
Receptor in rat diagonal band
*
Immunohistochemical staining of rat cortex frozen
sections using Anti- A1 Adenosine Receptor antibody
(#AAR-006), (1:100).
Immunohistochemical staining of rat cerebellum frozen
Immunohistochemical staining of rat diagonal band
sections using Anti-5-Hydroxytryptamine Receptor 1A
using Anti- A2A Adenosine Receptor antibody (#AAR-
antibody (#ASR-021), (1:100). 5-Hydroxytryptamine
002). A2A Adenosine Receptor expression (green)
Receptor 1A is expressed in Purkinje cells (red).
appears in the broca in individual neurons (arrows)
Hoechst 33342 is used as the counterstain.
and in neuropil (asterisk). Hematoxilin is used as
the counterstain.
1
2
3
A. A1 Adenosine Receptor (green) appears in neurons
(triangles) and in astrocytes (arrows).
B. Parvalbumin staining (red) appears in cortical
interneurons.
C. Confocal merge of images A and B demonstrates the
existence of A1 Adenosine Receptor in subset of cortical
interneurons and astrocytes.
4
95
72
55
36
Western blot analysis of rat brain
28
(lanes 1 and 3) and rat kidney
Western blot analysis of mouse (lanes 1 and 3) and rat
Western blot analysis of RAEC (lanes 1,3), rat
(lanes 2 and 4) lysates:
(lanes 2 and 4) brain membranes:
brain (lanes 2,4) and Jurkat (lanes 5,6) lysates:
1, 2. Anti-A1 Adenosine Receptor
1,2. Anti-5-Hydroxytryptamine Receptor 1A antibody
1,2,5. Anti-A2A Adenosine Receptor antibody
antibody (#AAR-006), (1:200).
(#ASR-021), (1:200).
(#AAR-002), (1:200).
3, 4. Anti-A1 Adenosine Receptor
3, 4. Anti-5-Hydroxytryptamine Receptor 1A antibody,
3,4,6. Anti-A2A Adenosine Receptor antibody,
antibody preincubated with the
preincubated with the control peptide antigen.
preincubated with the control peptide antigen.
control peptide antigen.
20
GPCR Pathways No.3 Winter 2010 www.alomone.com
Anti-A2B Adenosine Receptor
Anti-A3 Adenosine Receptor
(extracellular)
Cat. #: AAR-004
Cat. #: AAR-003
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Sizes: 50 µl | 0.2 ml
Epitope: Peptide (C)KETGAF YGREFKTAK, corresponding to amino acid residues 216-230
Host: Rabbit.
Epitope: Peptide KDSATNN*STEPWDGTTNESC,
corresponding to amino acid residues 147-166 of
of human A3 Adenosine Receptor (Accession P33765).
Applications: WB,IH, IC
human A2B Adenosine Receptor with replacement
of cysteine 154 (C154) with serine (*S) (Accession
P29275).
Applications: WB, IH
Expression of A3 Adenosine Receptor in rat lung
Expression of A2B Adenosine Receptor in rat lung
a
A
a
A
Br
A
Immunohistochemical staining of rat lung paraffin
embedded sections using Anti-A2B Adenosine Receptor
antibody (#AAR-003), (1:50). Staining is present in the
a
respiratory epithelium of the bronchiole (Br) as well as
in the pneumonocytes of the alveolar wall (alveoli, (A)).
Color reaction was obtained with SuperPicture
HRP-conjugated polymer (Zymed) followed by DAB.
Hematoxilin is used as the counterstain.
Immunohistochemical staining of paraffin-embedded sections of rat lung using Anti-A3 Adenosine
Receptor antibody (#AAR-004), (1:50). Staining is specific for respiratory epithelial cells of the
bronchiole (arrows) and pneumonocytes in the alveolar wall (a). Hematoxilin is used as the counterstain.
Expression of A3 Adenosine Receptor in human melanoma cell line
Western blot analysis of HL-60 cell line (lanes 1
A
B
C
D
E
F
and 2), rat brain (lanes 3 and 4) and mouse brain
(lanes 5 and 6) lysates:
1,3,5. Anti-A2B Adenosine Receptor
(extracellular) antibody (#AAR-003), (1:200).
2,4,6. Anti-A2B Adenosine Receptor
(extracellular) antibody, preincubated with the
control peptide antigen.
Immunocytochemical staining of A3 Adenosine Receptor in human melanoma cells (A2058).
Paraformaldehyde-fixed and permeabilized cells were stained as described.
A, D. Staining was done using Anti-A3 Adenosine Receptor antibody (#AAR-004), (1:100), (red), followed by goat-antirabbit-AlexaFluor-555 secondary antibody.
B, E. Nuclear fluorescence staining of cells using the membrane-permeable DNA dye Hoechst 33342 (blue).
C. Merged images of panels A and B.
F. Merged images of panels D and E.
GPCR Pathways No.3 Winter 2010 www.alomone.com
21
Name
Cat. #
Anti-α1B-Adrenoceptor (extracellular)
Cat. #: AAR-018
Adrenoceptors
Anti-α1A-Adrenoceptor (extracellular)
AAR-015
Sizes: 50 µl | 0.2 ml
Anti-α1B-Adrenoceptor (extracellular)
AAR-018
Host: Rabbit.
Anti-α1B-Adrenoceptor-ATTO-488
(extracellular)
AAR-018-AG
Anti-β2-Adrenoceptor (extracellular)
AAR-016
Anti-β3-Adrenoceptor (extracellular)
AAR-017
Epitope: Peptide (C)KNANFTGPNQTSSNS,
corresponding to amino acid residues 21-35 of human
α1B-Adrenoceptor (Accession P35368).
Applications: WB,IC, IFC
Expression of α1B-Adrenoceptor in living GH3 cells
Anti-α1B-Adrenoceptor-ATTO-488
(extracellular)
Cat. #: AAR-018-AG
Sizes: 50 µl
Host: Rabbit.
Epitope: Peptide (C)KNANFTGPNQTSSNS,
corresponding to amino acid residues 21-35 of human
α1B-Adrenoceptor (Accession P35368).
Applications: IC, FC
Immunocytochemical staining of α1B-Adrenoceptor expressing cells with Anti-α1B-Adrenoceptor
(extracellular) antibody (#AAR-018), (1:100), followed by goat-anti-rabbit-AlexaFluor-488
secondary antibody (green). Nuclear fluorescence staining of cells using the membranepermeable DNA dye Hoechst 33342 (blue).
Flow cytometry analysis of α1B-Adrenoceptor in
live intact GH3 cells
Indirect flow cytometry analysis of α1BAdrenoceptor in live intact GH3
1
2
3
4
250
130
5
6
95
72
250
130
95
72
55
55
36
28
36
28
Unstained GH3 cells.
Western blot analysis of rat brain (lanes 1and 3), rat kidney
GH3 cells + Anti-α1B-Adrenoceptor-ATTO-488
(lanes 2 and 4) and GH3 cell line (lanes 5 and 6) lysates:
(extracellular) antibody (#AAR-018-AG), (10µg/5x105 cells).
1,2,5. Anti-α1B-Adrenoceptor (extracellular) antibody (#AARUnstained GH3 cells.
GH3 cells + Anti-α1B-Adrenoceptor (extracellular)
(see IC picture on page 18)
22
antibody (#AAR-018), (10µg/5x105 cells).
018), (1:200).
3,4,6. Anti-α1B-Adrenoceptor (extracellular) antibody,
preincubated with the control peptide antigen.
GPCR Pathways No.3 Winter 2010 www.alomone.com
Anti-β3-Adrenoceptor (extracellular)
Anti-β2-Adrenoceptor (extracellular)
Anti-α1A-Adrenoceptor (extracellular)
Cat. #: AAR-017
Cat. #: AAR-016
Cat. #: AAR-015
Sizes: 50 µl | 0.2 ml
Sizes: 50 µl | 0.2 ml
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Host: Rabbit.
Host: Rabbit.
Epitope: Peptide DAEAQESH*SNPRC, corresponding
Epitope: Peptide (C)NGSRAPDHDVTQERDE,
Epitope: Peptide EDETI*SQINEEPG(C), corresponding
to amino acid residues 180-192 of mouse β3-
corresponding to amino acid residues 15-30 of mouse
to amino acid residues 171-183 of human α1A-
Adrenoceptor with replacement of cystein 186 (C186)
β2-Adrenoceptor (Accession P18762).
Adrenoceptor with replacement of cysteine 176 (C176)
with serine (*S) (Accession P25962 ).
Applications: WB, IH
with serine (*S) (Accession P35348).
Applications: WB, IH
Applications: WB, IH
Expression of β3-Adrenoceptor in rat hippocampus
Expression of β2-Adrenoceptor in rat lung
Expression of α1A-Adrenoceptor in rat cerebellum
A
B
C
H
Immunohistochemical staining of rat hippocampal dentate
Immunohistochemical staining of paraffin-embedded
gyrus using Anti-β3-Adrenoceptor (extracellular) antibody
sections of rat lung using Anti-β2-Adrenoceptor
(#AAR-017), (1:100), (green). β3-Adrenoceptor is strongly
(extracellular) antibody (#AAR-016), (1:100). β2-
Immunohistochemical staining of rat cerebellum using
expressed in the hilus (H) and in the outer molecular layer
Adrenoceptor is expressed in the respiratory epithelium.
Anti-α1A-Adrenoceptor (extracellular) antibody (#AAR-015),
(arrows). The distribution of β3-Adrenoceptor overlaps the
Hematoxilin is used as the counterstain.
(1:100).
entire layers, and not restricted to nerve cells (stained
A. α1A-Adrenoceptor (green) appears in fibers of Bergmann
red with mouse anti-parvalbumin). DAPI is used as the
glia (white triangle points at an example).
counterstain (blue).
B. S100beta (red), a marker of Bergmann glia, is stained in
the same section.
C. Merge of the images demonstrates expression of α1AAdrenoceptor in fibers of Bergmann glia. α1A-Adrenoceptor
also is expressed in cells in the molecular layer that are
s100beta negative (white arrow points at an example). DAPI
is used as the counterstain (blue).
1
2
3
1 2 3 4
4
150
100
75
250
150
100
75
50
50
37
37
25
25
20
Western blot analysis of PC3 cell line
1
2
3
4
lysate (lanes 1 and 3) and rat brain
250
150
100
75
Western blot analysis of mouse brain (lanes 1 and 3)
Western blot analysis of rat heart (lanes 1 and 3) and
and rat brain (lanes 2 and 4) lysates:
lung (lanes 2 and 4) lysates:
1, 3. Anti-β3-Adrenoceptor (extracellular) antibody
1, 3. Anti-β2-Adrenoceptor (extracellular) antibody
(#AAR-017), (1:200).
(#AAR-016), (1:200).
37
2, 4. Anti-β3-Adrenoceptor (extracellular) antibody,
2, 4. Anti-β2-Adrenoceptor (extracellular) antibody,
preincubated with the control peptide antigen.
preincubated with the control peptide antigen.
25
20
GPCR Pathways No.3 Winter 2010 www.alomone.com
50
membrane (lanes 2 and 4):
1, 2. Anti-α1A-Adrenoceptor
(extracellular) antibody (#AAR-015),
(1:200).
3, 4. Anti-α1A-Adrenoceptor
(extracellular) antibody, preincubated
with the control peptide antigen.
23
Name
Cat. #
Anti-Angiotensin-(1-7) Mas Receptor
Cat. #: AAR-013
Angiotensin-(1-7) Mas Receptor
Anti-Angiotensin-(1-7) Mas Receptor
AAR-013
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide (C)KIRKNTWASHSSK,
corresponding to amino acid residues 212-224 of rat
Angiotensin-(1-7) Mas Receptor (Accession P12526).
Applications: WB,IH
Expression of Angiotensin-(1-7) Mas Receptor in rat kidney
PT
G
G
G
A
CD
DT
CD
Immunohistochemical staining of paraffin embedded sections of rat kidney using AntiAngiotensin-(1-7) Mas Receptor antibody (#AAR-013), (1:100).
Angiotensin-(1-7) Mas Receptor is visualized (brown staining) in proximal tubules (PT) and distal
tubules (DT) in the renal cortex. Note that collecting ducts (CD) are less stained and both glomeruli
(G) and blood vessels (A) are negative. Hematoxilin is used as the counterstain.
1
2
3
4
5
6
75
50
37
25
20
1
(lanes 2 and 5) and heart (lanes 3 and 6) membranes:
1, 2, 3. Anti-Angiotensin-(1-7) Mas Receptor antibody (#AAR013), (1:200).
4, 5, 6. Anti-Angiotensin-(1-7) Mas Receptor antibody,
preincubated with the control peptide antigen.
1
2
35
72
Western blot analysis of human HeLa
55
1. Anti-Angiotensin-(1-7) Mas Receptor
36
28
cervix adenocarcinoma cell line lysate:
antibody (#AAR-013), (1:200).
2. Anti-Angiotensin-(1-7) Mas Receptor
antibody, preincubated with the control
peptide antigen.
24
Western blot analysis of rat brain (lanes 1 and 4) kidney
2
100
75
Western blot analysis of mouse kidney
50
1. Anti-Angiotensin-(1-7) Mas Receptor
37
25
20
membranes:
antibody (#AAR-013), (1:200).
2. Anti-Angiotensin-(1-7) Mas Receptor
antibody, preincubated with the control
peptide antigen.
GPCR Pathways No.3 Winter 2010 www.alomone.com
Name
Cat. #
Angiotensin II Receptors
Anti-Angiotensin II Receptor Type-1
(extracellular)
AAR-011
Anti-Angiotensin II Receptor Type-1
(extracellular)-ATTO-550
AAR-011-AO
Anti-Angiotensin II Receptor Type-2
(extracellular)
AAR-012
Anti-Angiotensin II Receptor Type-2
(extracellular)-ATTO-488
AAR-012-AG
Anti-Angiotensin II Receptor Type-2
Anti-Angiotensin II Receptor Type-1
(extracellular)
(extracellular)
Cat. #: AAR-012
Cat. #: AAR-011
Sizes: 50 µl | 0.2 ml
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Host: Rabbit.
Epitope: Peptide DNLNATGTNESAFNC, corresponding
Epitope: Peptide NSSTEDGIKRIQDDC, corresponding
to amino acid residues 21-35 of rat Angiotensin II
to amino acid residues 4-18 of human Angiotensin II
Receptor Type-2 (Accession P35351).
Receptor Type-1 (Accession P30556).
Applications: WB, IH, IC
Applications: WB, IH, IC, IFC
Expression of Angiotensin II Receptor Type-2 in
rat brain
Expression of Angiotensin II Receptor Type-1
in mouse cerebellum
A
B
3rd V
Optic
nerve
Immunohistochemical staining using Anti-Angiotensin II
Receptor Type-2 (extracellular) antibody (# AAR-012), (1:100)
in rat brain sections. Angiotensin II Receptor Type-2 is
Anti-Angiotensin II Receptor Type-1
stained red, and Nissl counterstain is in blue. A. Angiotensin
II Receptor Type-2 expressing neurons are scattered in the
(extracellular)-ATTO-550
paraventricular nucleus of the hypothalamus (arrows), and
Cat. #: AAR-011-AO
Receptor Type-2 is expressed in neurons in the supraoptic
in the vicinity of the 3rd ventricle (3rd V). B. Angiotensin II
nucleus (arrows), adjacent to the optic nerve.
Sizes: 50 µl
Host: Rabbit.
Epitope: Peptide NSSTEDGIKRIQDDC, corresponding
to amino acid residues 4-18 of human Angiotensin II
Receptor Type-1 (Accession P30556).
Applications: IH, IC
Expression of Angiotensin II Receptor Type-2 in
GH3 cells
Immunohistochemical staining of mouse
cerebellum using Anti-Angiotensin II Receptor
Type-1 (extracellular) antibody (#AAR-011), (1:100).
Angiotensin II Receptor Type-1 is present in the
(see figure on page 18)
Purkinje layer (green). Arrows point at Angiotensin
II Receptor Type-1 immunoreactive cells. Staining of
the same section with mouse anti-parvalbumin (red)
reveals partial co-localization.
Immunocytochemical staining of live intact rat GH3 pituitary
cells with Anti-Angiotensin II Receptor Type-2 (extracellular)
Anti-Angiotensin II Receptor Type-2
antibody (#AAR-012), (1:100), followed by goat-anti-rabbitAlexaFluor-555 secondary antibody.
(extracellular)-ATTO-488
Cat. #: AAR-012-AG
Western blot analysis of rat brain
Sizes: 50 µl
membranes:
Host: Rabbit.
1. Anti-Angiotensin II Receptor Type-
Epitope: Peptide DNLNATGTNESAFNC, corresponding
2 (extracellular) antibody, (#AAR-
Western blot analysis of rat liver (lanes 1 and 3) and rat
to amino acid residues 21-35 of rat Angiotensin II
012), (1:500).
kidney (lanes 2 and 4) membranes:
2. Anti-Angiotensin II Receptor
1, 2. Anti-Angiotensin II Receptor Type-1 (extracellular)
Type-2 (extracellular) antibody,
antibody (#AAR-011), (1:200).
preincubated with the control
3, 4. Anti-Angiotensin II Receptor Type-1 (extracellular)
peptide antigen.
antibody, preincubated with the control peptide antigen.
Receptor Type-2 (Accession P35351).
Applications: IH, IC
(see figure on page 18)
GPCR Pathways No.3 Winter 2010 www.alomone.com
25
Name
Cat. #
Anti-Bombesin Receptor 3 (extracellular)
Cat. #: ABR-003
Bombesin Receptors
Western blot analysis of rat brain
Anti-Bombesin Receptor 1
ABR-004
Anti-Bombesin Receptor 2
(extracellular)
ABR-002
Anti-Bombesin Receptor 3
(extracellular)
membranes:
Sizes: 50 µl | 0.2 ml
1. Anti-Bombesin Receptor 3
Host: Rabbit.
(extracellular) antibody (#ABR-003),
Epitope: Peptide (C)SNVYTFRDPNKNMTFE,
(1:200).
corresponding to amino acid residues 186-201 of
ABR-003
2. Anti-Bombesin Receptor 3
human Bombesin Receptor 3 (Accession P32247).
(extracellular) antibody, preincubated
Applications: WB, IH, IFC
with the control peptide antigen.
Expression of Bombesin Receptor 3 in rat testes
Immunohistochemical staining of Bombesin
Receptor 3 in rat testes. Paraffin embedded
sections of rat testes showing the area
of efferent ductules near the epididymis.
Intense stain (brown) is specific for the
Anti-Bombesin Receptor 1
pseudostratified epithelium of the efferent
Cat.#: ABR-004
After citrate treatment for antigen retrieval,
ductules.
slides were incubated overnight at 4°C with
Sizes: 50 µl | 0.2 ml
Anti-Bombesin Receptor 3 (extracellular)
Host: Rabbit.
for the color reaction. Hematoxilin and Eosin
antibody (#ABR-003), (1:50). DAB was used
Epitope: Peptide (C)KSAHNLPGEYNEHTKK,
are used as the counterstains.
corresponding to amino acid residues 241-256 of
human Bombesin Receptor 1 (Accession P28336).
Applications: WB, IH, IC
Anti-Bombesin Receptor 2 (extracellular)
Expression of Bombesin Receptor 1 in rat
hypothalamus
A
B
Cat. #: ABR-002
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide (C)RSYHYSEVDTSMLH, corresponding to amino acid
residues 287-300 of human Bombesin Receptor 2 (Accession P30550).
*
Applications: WB, IH, IC
Expression of Bombesin Receptor 2 in human colon cancer
C
A
B
C
D
*
Immunohistochemical staining of Bombesin Receptor 1 in rat
hypothalamus.
A. Frozen sections were stained with Anti-Bombesin
Receptor 1 antibody (#ABR-004), (1:200). Staining (red)
appears in the neuropil in the vicinity of the 3rd ventricle
(asterisk) and in neurons (triangles).
B. Calbindin D28k staining (green) appears in neurons.
Immunohistochemical staining of Bombesin Receptor 2 in human colon cancer sections. Longitudinal sections of paraffin-
C. Merge of (A) and (B) shows co-expression of Bombesin
embedded human colon showing malignant growth (A and B) and normal colon (C and D). Staining is specific for absorptive
Receptor 1 and Calbindin in a few neurons (vertical arrow).
epithelial cells in the crypts of Lieberkuhn (C and D) and for epithelium-derived malignant cells (A and B). Following antigen
DAPI is used as the counterstain (blue). The 3rd ventricle is
retrieval, slides were incubated overnight at 4°C with Anti-Bombesin Receptor 2 (extracellular) antibody (#ABR-002), (1:50).
indicated by a horizontal arrow.
Histofine (pink) was used for the color reaction. Hematoxilin is used as the counterstain.
26
GPCR Pathways No.3 Winter 2010 www.alomone.com
Name
Cat. #
Anti-B1 Bradykinin Receptor
Cat. #: ABR-011
Bradykinin Receptors
Anti-B1 Bradykinin Receptor
ABR-011
Sizes: 50 µl | 0.2 ml
Anti-B2 Bradykinin Receptor
ABR-012
Host: Rabbit.
Epitope: Peptide (C)KEASRTR*SGGPKGSK, corresponding to amino acid residues 243-257 of rat B1 Bradykinin
Receptor with replacement of cysteine 250 (C250) with serine (*S) (Accession P97583).
Applications: WB, IH
Expression of B1 Bradikinin Receptor in rat CNS
A
B
C
Western blot analysis of rat brain (1,3) and rat
heart (2,4) lysates:
1. Anti-B1 Bradykinin Receptor antibody
(#ABR-011), (1:200).
Immunohistochemical staining of rat brain frozen section (A and B) and cerebellum (C) using Anti-B1 Bradykinin Receptor
2. Anti-B1 Bradykinin Receptor antibody,
antibody (#ABR-011), (1:50). B1 Bradykinin Receptor is expressed in neuronal cells of the amygdala (C) and hippocampus (CA1)
preincubated with the control peptide antigen.
(B) as well as in the axons of Purkinje cells in the cerebellum (arrowheads on C). Hoechst 33342 is used as the counterstain.
Anti-B2 Bradykinin Receptor
Cat. #: ABR-012
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Expression of B2 Bradykinin Receptor in
mouse spinal cord
Epitope: Peptide GKRFRKKSWEVYQG(C),
corresponding to amino acid residues 336-349 of
A
B
human B2 Bradykinin Receptor (Accession P30411).
Applications: WB, IH
C
Immunohistochemical staining of mouse spinal cord
using Anti- B2 Bradykinin Receptor antibody (#ABR012), (1:100).
A. B2 Bradykinin Receptor (red) appears in several
spinal cord neurons (triangles).
Western blot analysis of rat brain lysate:
B. Parvalbumin (green) appears in neurons.
1. Anti-B2 Bradykinin Receptor antibody
C. merge of B2 Bradykinin Receptor and parvalbumin
(#ABR-012), (1:1000).
shows co-expression in several neurons (triangles)
2. Anti-B2 Bradykinin Receptor antibody,
but not all (arow).
preincubated with the control peptide antigen.
DAPI is used as the counterstain (blue).
GPCR Pathways No.3 Winter 2010 www.alomone.com
27
Name
Cat. #
Name
Calcium Sensing Receptor
Cat. #
Cannabinoid Receptors
Anti-Calcium Sensing Receptor
(extracellular)
ACR-004
Anti-Cannabinoid Receptor 2
ACR-002
Anti-Cannabinoid Receptor 2
(extracellular)
ACR-003
Anti-Calcium Sensing Receptor
(extracellular)
Cat. #: ACR-004
Anti-Cannabinoid Receptor 2
Sizes: 50 µl | 0.2 ml
Cat. #: ACR-002
Host: Rabbit.
Epitope: Peptide (C)DDYGRPGIEKFREE,
corresponding to amino acid residues
216-229 of human Calcium Sensing
Receptor (Accession P41180).
Applications: WB, IC, IFC
Western blot analysis of Colo205 (lanes 1and 3), rat liver
(lanes 2 and 4) and mouse kidney (lanes 5 and 6) lysates:
Sizes: 50 µl | 0.2 ml
1,2,5. Anti-Calcium Sensing Receptor (extracellular)
Host: Rabbit.
antibody (#ACR-004), (1:200).
Epitope: Peptide (C)DRQVPGIARMRLDVR,
3,4,6. Anti-Calcium Sensing Receptor (extracellular)
corresponding to amino acid residues 228-242 of
antibody, preincubated with the control peptide antigen.
rat Cannabinoid Receptor 2 (Accession Q9QZN9).
Applications: WB
Expression of Calcium Sensing Receptor in C6 cells
A
B
C
Western blot analysis of RBL (lanes 1,3), C6
(lanes 2,4) and rat lung (lanes 5,6) lysates:
1,2,5. Anti-Cannabinoid Receptor 2 antibody
(#ACR-002), (1:200).
Immunocytochemical staining of Calcium Sensing Receptor in live intact C6 cells (rat brain glioma).
3,4,6. Anti-Cannabinoid Receptor 2 antibody,
Paraformaldehyde-fixed and permeabilized C6 cells cells were stained as described.
preincubated with the control peptide antigen.
A. Staining was done using Anti-Calcium Sensing Receptor (extracellular) antibody (#ACR-004), (1:50) followed by goat-antirabbit-AlexaFluor-555 secondary antibody.
B. Nuclear fluorescence staining of cells using the membrane-permeable DNA dye Hoechst 33342.
C. Merged images of panels A and B.
Indirect flow cytometry analysis of Calcium Sensing Receptor in live Jurkat cells
Unstained Jurkat cells
Jurkat cells + Anti-Calcium Sensing Receptor
(extracellular) antibody (#ACR-004), (10µg per 5x105 cells).
28
GPCR Pathways No.3 Winter 2010 www.alomone.com
Anti-Cannabinoid Receptor 2 (extracellular)
Name
Cat. #: ACR-003
Cat. #
Cholecystokinin Receptors
Sizes: 50 µl | 0.2 ml
Anti-Cholecystokinin B Receptor
(extracellular)
Host: Rabbit.
ACR-042
Epitope: Peptide (C)NGSKDGLDSNPMKD, corresponding to residues
11-24 of human Cannabinoid Receptor 2 (Accession P34972).
Applications: WB, IC, IFC
Anti-Cholecystokinin B Receptor
(extracellular)
Expression of Cannabinoid Receptor 2 in human prostate carcinoma cells
Cat. #: ACR-042
A
Sizes: 50 µl | 0.2 ml
B
C
Host: Rabbit.
Epitope: Peptide CETPRIRGTGTRELE corresponding to
amino acid residues 39-53 of mouse Cholecystokinin
B receptor (Accession P56481).
Applications: WB, IH
A. Immunocytochemical staining of LNCaP cells with Anti-Cannabinoid Receptor 2 (extracellular) antibody (#ACR-003), (1:50),
followed by goat-anti-rabbit-AlexaFluor-488 secondary antibody.
B. Nuclear staining of LNCaP cells with the cell permeable dye Hoechst 33342.
C. Live intact LNCaP cells.
Expression of Cholecystokinin B Receptor in rat
stomach
Indirect flow cytometry analysis of Cannabinoid Receptor 2 in live intact HL-60 cells
Immunohistochemical staining of paraffin embedded
sections of rat stomach using Anti-Cholecystokinin B
Receptor (extracellular) antibody (#ACR-042), (1:100).
HL-60 cells + goat-anti-rabbit-FITC.
HL-60 cells + Anti-Cannabinoid Receptor 2
(extracellular) antibody (#ACR-003), (5 µg) + goatanti-rabbit-FITC.
Cholecystokinin B Receptor (brown staining) is expressed
in both parietal cells (black arrows) and in chief cells (red
arrows) of the gastric mucosa. Hematoxilin is used as the
counterstain.
1
2
3
4
130
95
72
55
36
28
Western blot analysis of HL-60 (lanes 1,2) and MCF-7 (lanes
Western blot analysis of mouse (lanes 1 and 3) and
3,4) cell line lysates:
rat (lanes 2 and 4) brain membranes:
1,3. Anti-Cannabinoid Receptor 2 (extracellular) antibody
1, 2. Anti-Cholecystokinin B Receptor (extracellular)
(#ACR-003), (1:200).
antibody (#ACR-042), (1:200).
2,4. Anti-Cannabinoid Receptor 2 (extracellular) antibody,
3, 4. Anti-Cholecystokinin B Receptor (extracellular),
preincubated with the control peptide antigen.
preincubated with the control peptide antigen.
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Name
Cat. #
Dopamine Receptors
Anti-D2 Dopamine Receptor
(extracellular)
ADR-002
Anti-D3 Dopamine Receptor
(extracellular)
ADR-003
Anti-D5 Dopamine Receptor
(extracellular)
ADR-005
Anti-D2 Dopamine Receptor
(extracellular)
Anti-D3 Dopamine Receptor
Anti-D5 Dopamine Receptor
(extracellular)
(extracellular)
Cat. #: ADR-003
Cat. #: ADR-005
Sizes: 50 µl | 0.2 ml
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Host: Rabbit.
Epitope: Peptide CGAENSTGVNRARPH, corresponding
Epitope: EEGWELEGRTENC, corresponding to amino
to amino acid residues 15-29 of rat D3 Dopamine
acid residues 199-211 of rat D5 Dopamine Receptor
Receptor (Accession P19020).
(Accession P25115).
Applications: WB,IH
Applications: WB,IH
Expression of D3 Dopamine Receptor in rat
striatum
Expression of D5 Dopamine Receptor in rat
striatum
A
A
B
B
Cat. #: ADR-002
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide (C)DDLERQNWSRPFNGSE,
corresponding to amino acid residues 11-26 of
rat D2 Dopamine Receptor (Accession P61169).
C
C
Immunohistochemical staining of perfusion-fixed frozen
Immunohistochemical staining of perfusion-fixed
Applications: WB
Expression of D2 Dopamine Receptor
1
2
3
4
5
6
7
8
130
95
72
55
36
28
rat brain sections with Anti-D3 Dopamine Receptor
frozen brain sections with Anti-D5 Dopamine Receptor
(extracellular) antibody (#ADR-003), (1:100).
(extracellular) antibody (#ADR-005), (1:100).
A. D3 Dopamine Receptor appears in a subset of striatal
A. D5 Dopamine Receptor appears in a subset of striatal
Western blot analysis of rat striatum (lanes 1 and 5) and
neurons (green fluorescence).
neurons and in the striatal matrix (green fluorescence).
hippocampus (lanes 2 and 6) membranes and of rat (lanes
B. The same section was stained for parvalbumin (red
B. Staining of the same section with calbindin D28k (red
3 and 7) and mouse (lanes 4 and 8) whole brain lysates:
fluorescence), a marker of interneurons.
fluorescence), a marker of interneurons.
1- 4. Anti-D2 Dopamine Receptor (extracellular) antibody
C. Merged images of panels A and B. D3 Dopamine
C. Merged images of panels A and B. D5 Dopamine
(#ADR-002), (1:200).
Receptor localization includes parvalbuminergic striatal
Receptor partially overlaps with the population of
5- 8. Anti-D2 Dopamine Receptor (extracellular) antibody,
interneurons. Nuclei were stained with DAPI (blue) as the
calbindin containing striatal interneurons.
preincubated with the control peptide antigen.
counterstain.
Nuclei were stained with DAPI (blue) as the counterstain.
1
2
1
130
95
72
55
36
28
30
2
150
100
75
50
37
Western blot analysis of mouse brain membranes:
Western blot analysis of rat striatum membranes:
1. Anti-D3 Dopamine Receptor (extracellular) antibody
1. Anti-D5 Dopamine Receptor (extracellular) antibody
(#ADR-003), (1:200).
(#ADR-005), (1:200).
2. Anti-D3 Dopamine Receptor (extracellular) antibody,
2. Anti-D5 Dopamine Receptor (extracellular) antibody,
preincubated with the control peptide antigen.
preincubated with the control peptide antigen.
GPCR Pathways No.3 Winter 2010 www.alomone.com
Name
Cat. #
Anti-Galanin Receptor Type 1
Cat. #: AGR-011
Galanin Receptors
1
Anti-Galanin Receptor Type 1
AGR-011
Anti-Galanin Receptor Type 3
(extracellular)
AGR-013
250
150
100
75
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide RSKPGKPRSTTN(C), corresponding
to amino acid residues 60-71 of mouse Galanin
Receptor Type 1 (Accession P56479).
50
37
25
20
Applications: WB, IH, IC
Anti-Galanin Receptor Type 3
2
Western blot analysis of rat brain lysate:
1. Anti-Galanin Receptor Type 1
antibody (#AGR-011), (1:200).
2. Anti-Galanin Receptor Type 1
antibody, preincubated with the control
peptide antigen.
Expression of Galanin Receptor Type 1 in rat DRG
(extracellular)
Cat. #: AGR-013
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide SYYGTVRYGALELC, corresponding
to amino acid residues 159-172 of mouse Galanin
Receptor Type 3 (Accession O88853).
Applications: WB
Immunohistochemical staining of
rat dorsal root ganglia (DRG) frozen
sections using Anti-Galanin Receptor
Type 1 antibody (#AGR-011), (1:200),
(red).
Galanin Receptor Type 1 is expressed in
Expression of Galanin Receptor Type 3
DRG neuron cells (red). Hoechst 33342
1
is used as the counterstain (blue).
2
130
95
72
55
36
28
17
Western blot analysis of Galanin Receptor Type 3 in rat
brain membrane:
Expression of Galanin Receptor Type 1 in rat DRG
A
B
C
D
E
F
1. Anti-Galanin Receptor Type 3 (extracellular) antibody
(#AGR-013),(1:200).
2. Anti-Galanin Receptor Type 3 (extracellular) antibody,
preincubated with the control peptide antigen.
Immunocytochemical staining of Galanin Receptor Type 1 in primary cultures of rat dorsal root ganglion (DRG) neurons.
A, D. Paraformaldehyde-fixed and permeabilized DRG primary culture was stained with Anti-Galanin Receptor Type 1 antibody
(#AGR-011), (1:100), followed by goat-anti-rabbit-AlexaFluor-555 secondary antibody (red).
B, E. Nuclear fluorescence staining of cells using the membrane-permeable DNA dye Hoechst 33342 (blue).
C. Merged images of panels A and B.
F. Merged images of panels D and E.
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Name
Cat. #
Anti-mGluR5 (extracellular)
Cat. #: AGC-007
Glutamate Receptors
Anti-mGluR1 (extracellular)
AGC-006
Sizes: 50 µl | 0.2 ml
Anti-mGluR2 (extracellular)
AGC-011
Host: Rabbit.
Anti-mGluR3 (extracellular)
AGC-012
Anti-mGluR5 (extracellular)
AGC-007
Western blot analysis of rat brain
membranes:
1. Anti-mGluR5 (extracellular) antibody
Epitope: Peptide EGFAQENSKYNKTC,
(#AGC-007), (1:500).
corresponding to amino acid residues 367-380
2. Anti-mGluR5 (extracellular) antibody,
of rat mGluR5 (Accession P31424).
preincubated with the control peptide
Applications: WB, IH, IC
antigen.
Anti-mGluR3 (extracellular)
Cat. #: AGC-012
Expression of mGluR5 in rat hippocampus
Expression of mGluR5 in rat cerebellum
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Molec.
Epitope: Peptide (C)GDHNFMRREIKIEGD,
corresponding to amino acids 24-38 of rat mGluR3
(Accession P31422).
Applications: WB, IH
Granule
Molec.
Expression of mGluR3 in rat brain stem sections
Granule
Immunohistochemical staining of frozen brain stem
sections with Anti-mGluR3 (extracellular) antibody
(#AGC-012), (1:100), followed by goat-anti-rabbitAlexaFluor-555 secondary antibody (red). Staining is
present in neuronal cell bodies (arrows) in the brainstem
Immunohistochemical staining of perfusion-fixed
Immunohistochemical staining of perfusion-fixed frozen
frozen sections of rat hippocampus with Anti-mGluR5
sections of rat cerebellum with Anti-mGluR5 (extracellular)
(extracellular) antibody (#AGC-007), (1:50). mGluR5 (red)
antibody (#AGC-007), (1:50). mGluR5 (red) was detected in
was detected in CA3 cells (triangles). Staining with mouse
cerebellar Purkinje cells (arrows) and in the molecular layer
anti-parvalbumin (green) revealed co-localization in
(triangles). Staining with mouse-anti-parvalbumin (green)
pyramidal layer. DAPI counterstain was used to visualize
revealed co-localization in Purkinje but not in the molecular
nuclei of all cells (blue).
layer. Little staining of mGluR5 was detected in the granule
layer. DAPI counterstain was used to visualize nuclei of all
nuclei. Hoechst 33342 (blue) is used as the counterstain.
1
2
3
cells (blue).
4
150
100
Expression of mGluR5 in rat GH3 cells
75
50
Western blot analysis of rat (lanes 1 and 3) and mouse (lanes
Immunocytochemical staining of live
2 and 4) brain membranes:
intact rat GH3 pituitary cells with
1, 2. Anti-mGluR3 (extracellular) antibody (#AGC-012),
Anti-mGluR5 (extracellular) antibody
(1:600).
(#AGC-007), (1:100), followed by
3, 4. Anti-mGluR3 (extracellular) antibody, preincubated with
goat-anti-rabbit-AlexaFluor-555
the control peptide antigen.
secondary antibody.
32
GPCR Pathways No.3 Winter 2010 www.alomone.com
Anti-mGluR1 (extracellular)
Anti-mGluR2 (extracellular)
Cat. #: AGC-006
Cat. #: AGC-011
Sizes: 50 µl | 0.2 ml
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Host: Rabbit.
Epitope: Peptide (C)HEGVLNIDDYKIQMNK,
Epitope: Peptide SLSRGADGSRHIC, corresponding
corresponding to amino acid residues 501-516 of rat
to amino acids 109-121 of rat mGluR2 (Accession
mGluR1 (Accession P23385).
P31421).
Applications: WB, IH, IFC, IC, IP
Applications: WB, IH
Expression of mGluR1 in rat C6 cells
Expression of mGluR2 in rat brain
B
A
CC
CC
OR
OR
C
CC
Immunocytochemical staining of live intact rat C6 glioma cells with Anti-mGluR1 (extracellular) antibody
(#AGC-006), (1:100), followed by goat-anti-rabbit-AlexaFluor-555 secondary antibody (red). Nuclei were
stained with the cell permeable dye Hoechst 33342 (blue staining).
OR
Indirect flow cytometry analysis of mGluR1
in Jurkat cells
Immunohistochemical staining of perfusion-fixed frozen
brain sections with Anti-mGluR2 (extracellular) antibody
(#AGC-011), (1:100).
A. mGluR2 (green fluorescence) is visualized in the corpus
callosum (CC) and hippocampal stratum oriens (OR).
B. Glial fibrillary acidic protein (GFAP) (red fluorescence), a
Immunoprecipitation of rat brain lysate with Anti-
marker of astrocytes.
mGluR1 (extracellular) antibody (#AGC-006)(5 µg):
C. Merge of the two images demonstrates expression of
1. Cell lysate + protein A beads + Anti-mGluR1
mGluR2 in astrocytes.
(extracellular) antibody.
DAPI is used as the nuclear counterstain (blue).
2. Cell lysate + protein A beads + pre-immune rabbit
serum.
3. Cell lysate.
Black arrow indicates the mGluR1 protein (glycosylated
Jurkat cells + goat-anti-rabbit-FITC.
form) while the red arrow shows the IgG heavy chain.
Jurkat cells + Anti-mGluR1 (extracellular) antibody
Immunoblot was performed with the Anti-mGluR1
(#AGC-006), (10 µg) + goat-anti-rabbit-FITC.
(extracellular) antibody.
1
2
3
4
250
130
95
72
55
Western blot analysis of rat brain
Western blot analysis of mouse
lysate:
brain lysate:
Western blot analysis of rat cerebellum (lanes 1 and 3)
1. Anti-mGluR1 (extracellular)
1. Anti-mGluR1 (extracellular)
and cortex (lanes 2 and 4) membranes:
antibody (#AGC-006), (1:200).
antibody (#AGC-006), (1:200).
1, 2. Anti-mGluR2 (extracellular) antibody (#AGC-011),
2. Anti-mGluR1 (extracellular)
2. Anti-mGluR1 (extracellular)
(1:400).
antibody, preincubated with the
antibody, preincubated with the
3, 4. Anti-mGluR2 (extracellular) antibody, preincubated
control peptide antigen.
control peptide antigen.
with the control peptide antigen.
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33
Name
Cat. #
Anti-Rat H2 Histamine Receptor (extracellular)
Cat. #: AHR-002
Histamine Receptors
Anti-H1 Histamine Receptor
AHR-001
Anti-Rat H2 Histamine Receptor
(extracellular)
AHR-002
Anti-H3 Histamine Receptor
AHR-003
Anti-Human H4 Histamine Receptor
(extracellular)
AHR-004
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide RNGTRGGNDTFKC, corresponding to amino
acids 161-173 of rat H2 Histamine Receptor (Accession P25102).
Applications: WB,IH, IFC
Expression of H2 Histamine Receptor in rat stomach
Immunohistochemical staining of
paraffin embedded sections of rat
stomach using Anti-Rat H2 Histamine
Receptor (extracellular) antibody
(#AHR-002), (1:100). H2 Histamine
Receptor (brown staining) is expressed
in parietal cells (arrows) of the gastric
glands. Hematoxilin is used as the
counterstain .
Indirect flow cytometry analysis of H2 Histamine Receptor in live intact RBL cells
RBL cells + goat-anti-rabbit-FITC.
RBL Cells+ Anti-Rat H2 Histamine Receptor (extracellular)
antibody (#AHR-002), (1:20) + goat-anti-rabbit-FITC.
1
2
3
4
100
75
50
37
25
20
Western blot analysis of rat basophilic leukemia cells (RBL)
(lanes 1 and 3) and rat brain lysates (lanes 2 and 4):
1, 2. Anti-Rat H2 Histamine Receptor (extracellular) antibody
(#AHR-002), (1:200).
3, 4. Anti-Rat H2 Histamine Receptor (extracellular) antibody,
preincubated with the control peptide antigen.
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GPCR Pathways No.3 Winter 2010 www.alomone.com
Anti-H1 Histamine Receptor
Anti-H3 Histamine Receptor
Anti-Human H4 Histamine Receptor
Cat. #: AHR-001
Cat. #: AHR-003
(extracellular)
Sizes: 50 µl | 0.2 ml
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Host: Rabbit.
Epitope: Peptide (C)HSRQYVSGLHLNRE,
Epitope: Peptide (C)RTRLRLDGGREAGPE,
Cat. #: AHR-004
corresponding to amino acids 396-409 of rat H1
corresponding to amino acids 228-242 of rat H3
Histamine Receptor (Accession P31390).
Histamine Receptor (Accession Q9QYN8).
Applications: WB,IH
Applications: WB,IH
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide HTLFEWDFGKEIC, corresponding to
amino acids 75-87 of human H4 Histamine Receptor
(Accession Q9H3N8).
Applications: WB, IFC
Expression of H1 Histamine Receptor in mouse
brain
Expression of H3 Histamine Receptor in rat
cerebellum
A
A
B
Indirect flow cytometry analysis of H4 Histamine
Receptor in live intact Jurkat cells
B
C
Immunohistochemical staining of H1 Histamine Receptor
in mouse ventromedial hypothalamus (VMH) with Anti-H1
Histamine Receptor antibody (#AHR-001).
A. H1 Histamine Receptor (green fluorescence) appears in
the outline of the VMH nucleus (arrows).
B. DAPI (blue) counterstain labels all cell nuclei.
1
Jurkat cells + goat-anti-rabbit-PE.
2
Jurkat Cells+ Anti-Human H4 Histamine Receptor
150
100
75
(extracellular) antibody (#AHR-004), (1:20) + goat-antiImmunohistochemical staining of rat brain frozen
50
sections with Anti-H3 Histamine Receptor antibody
37
(#AHR-003), (1:100).
25
dendrites of Purkinje cells (arrows, green fluorescence).
rabbit-PE.
A. H3 Histamine Receptor was particularly expressed in
Western blot analysis of mouse brain membranes:
1. Anti-H1 Histamine Receptor antibody (#AHR-001), (1:400).
2. Anti-H1 Histamine Receptor antibody, preincubated with
the control peptide antigen.
B. Staining with mouse anti-parvalbumin (red
fluorescence) was detected in Purkinje cells and
interneurons in the molecular layer.
C. Merge of the two images demonstrates that the
staining was restricted to dendrites of Purkinje cells.
DAPI (blue) counterstain labels all cell nuclei.
1
2
3
4
130
95
72
55
33
28
1
2
3
4
100
75
1
2
3
Western blot analysis of human chronic myelogenous
4
150
leukemia (K562) (lanes 1 and 3) and human promyelocytic
50
95
72
leukemia (HL-60) (lanes 2 and 4) cell lysates:
37
55
antibody (#AHR-004), (1:400).
36
antibody, preincubated with the control peptide antigen.
1, 2. Anti-Human H4 Histamine Receptor (extracellular)
3, 4. Anti-Human H4 Histamine Receptor (extracellular)
25
Western blot analysis of rat heart membranes
(lanes 1 and 3) and rat basophilic leukemia
Western blot analysis of rat (lanes 1 and 3) and mouse
(RBL) cell lysates (lanes 2 and 4):
brain membranes (lanes 2 and 4):
1, 2. Anti-H1 Histamine Receptor antibody
1, 2. Anti-H3 Histamine Receptor antibody (#AHR-003),
(#AHR-001), (1:200).
(1:200).
3, 4. Anti-H1 Histamine Receptor antibody,
3, 4. Anti-H3 Histamine Receptor antibody, preincubated
preincubated with the control peptide antigen.
with the control peptide antigen.
GPCR Pathways No.3 Winter 2010 www.alomone.com
35
Name
Cat. #
Name
Kisspeptins Receptor
Cat. #
Melanocortin Receptors
Anti-Kisspeptins Receptor
(extracellular)
AKR-001
Anti-Melanocortin Receptor 1
AMR-020
Anti-Melanocortin Receptor 2
(extracellular)
AMR-022
Anti-Melanocortin Receptor 3
(extracellular)
AMR-023
Anti-Melanocortin Receptor 4
(extracellular)
AMR-024
Anti-Melanocortin Receptor 5
AMR-025
Anti-Kisspeptins Receptor (extracellular)
Cat. #: AKR-001
Anti-Melanocortin Receptor 3
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide (C)GSWHPRSYAAYALK, corresponding to amino acid
(extracellular)
residues 292-305 of human Kisspeptins Receptor (Accession Q969F8).
Cat. #: AMR-023
Applications: WB, IH
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide (C)NSDSLTLEDQFIQHMD,
corresponding to amino acid residues 102-117 of rat
Expression of Kisspeptins Receptor in rat
pancreas
Melanocortin Receptor 3 (Accession P32244).
Applications: WB, IH, IFC
Expression of Melanocortin Receptor 3 in rat
brain sections
Expression of Kisspeptins Receptor in rat
cerebellum
IL
A
B
*
Immunohistochemical staining of paraffin embedded
section of rat pancreas using Anti-Kisspeptins Receptor
(extracellular) antibody (#AKR-001), (1:100). Kisspeptins
C
Receptor staining (brown) appears in the Isles of
Langerhans (IL). Hematoxilin is used as the counterstain.
Immunohistochemical staining of perfusion-fixed
frozen brain sections with Anti-Melanocortin Receptor 3
(extracellular) antibody (#AMR-023), (1:100). Expression
1
2
3
4
5
7
6
(green fluorescence) appears in the hypothalamic
8
150
100
75
150
50
75
37
50
25
periventricular region (horizontal arrow), in some nerve
cells (vertical arrow), and in the neuropil (asterisk). Hoechst
100
33342 (blue) is used as the counterstain.
Western blot analysis of mouse
37
Immunohistochemical staining of rat cerebellum frozen
(lanes 1 and 3) and rat (lanes 2
sections using Anti-Kisspeptins Receptor (extracellular)
and 4) brain lysates:
Western blot analysis of jurkat (lanes 1 and 4), HL-60 (lanes
antibody (#AKR-001), (1:100).
1, 2. Anti-Melanocortin Receptor
2 and 5), MCF-7 (lanes 3 and 6) and rat brain lysates (lanes
A. Kisspeptins Receptor (green) is expressed particularly
3 (extracellular) antibody (#AMR-
7 and 8):
in Purkinje cell bodies (arrows).
023), (1:200).
1,2,3,7. Anti-Kisspeptins Receptor (extracellular) antibody
B. Staining with mouse anti-parvalbumin (red) is detected
3, 4. Anti-Melanocortin Receptor
(#AKR-001), (1:500).
in Purkinje cells and interneurons in the molecular layer.
3 (extracellular) antibody,
4,5,6,8. Anti-Kisspeptins Receptor (extracellular) antibody,
C. Merge of the two images demonstrates that the staining
preincubated with the control
preincubated with the control peptide antigen.
is restricted to Purkinje cells.
peptide antigen.
36
GPCR Pathways No.3 Winter 2010 www.alomone.com
Anti-Melanocortin Receptor 2
Anti-Melanocortin Receptor 4
Anti-Melanocortin Receptor 5
(extracellular)
(extracellular)
Cat. #: AMR-025
Cat. #: AMR-022
Cat. #: AMR-024
Sizes: 50 µl | 0.2 ml
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Host: Rabbit.
Epitope: Peptide (C)RNMGYLKPRGSFE, corresponding
Epitope: Peptide (C)HRGMHTSLHLWNRSS,
to amino acid residues 87-99 of human Melanocortin
corresponding to amino acid residues 6-20 of human
Receptor 2 (Accession Q01718).
Melanocortin Receptor 4 (Accession P32245 ).
Applications: WB, IH
Applications: WB, IH, IC
Expression of Melanocortin Receptor 2 in rat
adrenal gland
Expression of Melanocortin Receptor 4 in mouse
brain sections.
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide (C)RYNSVRQRTSMKG corresponding
to amino acid residues 224-236 of mouse
Melanocortin Receptor 5 (Accession P41149).
Applications: WB, IH
Expression of Melanocortin Receptor 5
1
2
3
4
95
72
ZF
ZG
C
55
36
28
Western blot analysis of rat (lanes 1 and 3) and
mouse (lanes 2 and 4) brain membranes:
1, 2. Anti-Melanocortin Receptor 5 antibody
(#AMR-025), (1:200).
3, 4. Anti-Melanocortin Receptor 5 antibody,
preincubated with the control peptide antigen.
Immunohistochemical staining of paraffin embedded
sections of rat adrenal cortex showing the capsule (C), the
zona glomerulosa (ZG) and the zona fasciculata (ZF) of the
adrenal cortex. Note that the staining (brown) is specific
Immunohistochemical staining of mouse brain. Perfusion-
for cells of the adrenal gland while no staining is present
fixed frozen sections were incubated with Anti-Melanocortin
in the dense connective tissue of the capsule. Following
Receptor 4 (extracellular) antibody (#AMR-024), (1:100).
antigen retrieval, slides were incubated overnight at 4°C
Melanocortin Receptor 4 expression (green fluorescence)
with Anti-Melanocortin Receptor 2 (extracellular) antibody
appears in the mouse hypothalamus in axonal processes
(#AMR-022), (1:100). Color reaction was obtained with DAB.
(arrows). Hoechst 33342 (blue) is used as the counterstain.
Hematoxilin and Eosin are used as the counterstains.
Anti-Melanocortin Receptor 1
Cat. #: AMR-020
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Immunohistochemical staining of Melanocortin Receptor 1 in normal skin and melanoma
Epitope: Peptide (C)HAQGIARLHKRQRPVH,
corresponding to amino acid residues 217-232 of
human Melanocortin Receptor 1 (Accession Q01726).
A
B
C
Applications: WB, IH, IC
Western blot analysis of rat
adrenal lysate:
1. Anti-Melanocortin Receptor 1
antibody (#AMR-020), (1:400).
Immunohistochemical staining of paraffin embedded sections of normal epidermis (A), normal hypodermis (B) and infiltrating
2. Anti-Melanocortin Receptor 1
melanoma (C) show highly specific staining (red-brown color) for Melanocortin Receptor 1. Following antigen retrieval, slides
antibody, preincubated with the
were incubated overnight at 4°C with Anti-Melanocortin Receptor 1 antibody (#AMR-020), (1:100). Color reaction was obtained
control peptide antigen.
with DAB. Hematoxilin is used as the counterstain.
GPCR Pathways No.3 Winter 2010 www.alomone.com
37
Name
Cat. #
Anti-Neuropeptide Y5 Receptor
Cat. #: ANR-025
Neuropeptide Y Receptors
Expression of Neuropeptide Y5 Receptor in
rat striatum
A
Anti-Neuropeptide Y1 Receptor
ANR-021
Sizes: 50 µl | 0.2 ml
Anti-Neuropeptide Y2 Receptor
ANR-022
Host: Rabbit.
Anti-Neuropeptide Y4 Receptor
ANR-024
Anti-Neuropeptide Y5 Receptor
ANR-025
B
*
Epitope: Peptide SFIRKHRRRYSKKTAC,
corresponding to amino acid residues 291-306 of
rat Neuropeptide Y5 Receptor (Accession Q63634 )
Applications: WB, IH, IC
1
C
2
250
150
100
75
50
Immunohistochemical staining of rat striatum using Anti-
37
Neuropeptide Y5 Receptor antibody (#ANR-025), (1:100).
A. Neuropeptide Y5 Receptor (red) appears in the striatal
25
matrix (asterisk) and in nerve cells in the matrix (vertical
Anti-Neuropeptide Y2 Receptor
Cat. #: ANR-022
Western blot analysis of rat brain membrane:
arrows).
1. Anti-Neuropeptide Y5 Receptor antibody
B. Parvalbumin (green) appears in the striatal matrix.
(#ANR-025), (1:200).
C. Merge of Neuropeptide Y5 Receptor and parvalbumin
2. Anti-Neuropeptide Y5 Receptor antibody,
demonstrates co-localization in a sub-set of cells
preincubated with the control peptide antigen.
(horizontal arrows). DAPI is used as the counterstain (blue).
Expression of Neuropeptide Y2 Receptor in rat DRG
A
B
C
D
E
F
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide CEQRLDAIHSEVSMTFKAK,
corresponding to amino acid residues 346364 of mouse Anti-Neuropeptide Y2 Receptor
(Accession P97295).
Applications: WB, IC
1
2
3
4
250
150
100
75
50
37
25
Immunocytochemical staining of Neuropeptide Y2 Receptor in a primary culture of rat dorsal root ganglion (DRG) neurons.
Western blot analysis of rat hippocampus and rat
A, D. Paraformaldehyde-fixed and permeabilized DRG primary culture stained with Anti-Neuropeptide Y2 Receptor antibody
whole brain lysates:
(#ANR-022), (1:100), followed by goat-anti-rabbit-AlexaFluor-555 secondary antibody.
1, 2. Anti-Neuropeptide Y2 Receptor antibody
B, E. Nuclear fluorescence staining of cells using the membrane-permeable DNA dye Hoechst 33342.
(#ANR-022), (1:200).
C. Merged images of panels A and B.
3, 4. Anti-Neuropeptide Y2 Receptor antibody,
F. Merged images of panels D and E.
preincubated with the control peptide antigen.
Magnification: A-C: x20 E-F: x100
38
GPCR Pathways No.3 Winter 2010 www.alomone.com
Anti-Neuropeptide Y4 Receptor
Cat. #: ANR-024
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide NINFKKDIKALVLTC, corresponding
to amino acid residues 326-340 of rat
Neuropeptide Y4 Receptor (Accession Q63447).
Applications: WB, IC
1
2
3
4
5
6
Expression of Neuropeptide Y4 Receptor in rat DRG
A
B
C
D
E
F
250
150
100
75
50
37
Western blot analysis of rat brain membrane (lanes
Immunocytochemical staining of Neuropeptide Y4 Receptor in a primary culture of rat dorsal root ganglia (DRG) neurons.
1 and 4), cortex (lanes 2 and 5), and hippocampus
A, D. Paraformaldehyde-fixed and permeabilized DRG primary culture stained with Anti-Neuropeptide Y4 Receptor
(lanes 3 and 6) lysates:
antibody (#ANR-024), (1:100), followed by goat-anti-rabbit-AlexaFluor-488 secondary antibody (green).
1, 2, 3. Anti-Neuropeptide Y4 Receptor antibody
B, E. Nuclear fluorescence staining of cells using the membrane-permeable DNA dye Hoechst 33342 (blue).
(#ANR-024), (1:200).
C. Merged images of panels A and B.
4, 5, 6. Anti-Neuropeptide Y4 Receptor antibody
F. Merged images of panels D and E.
preincubated with the control peptide antigen.
Magnification:
A-C: x20.
E-F: x100.
Anti-Neuropeptide Y1 Receptor
Cat. #: ANR-021
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide (C)RLKRRNNMMDKMRDNK,
Expression of Neuropeptide Y1 Receptor in
rat striatum
A
B
corresponding to amino acid residues 237-252 of
human Neuropeptide Y1 Receptor (Accession P25929).
Applications: WB, IH, IC
*
C
Immunohistochemical staining of rat striatum using AntiWestern blot analysis of Jurkat (lanes 1 and 4), K562
Neuropeptide Y1 Receptor antibody (#ANR-021), (1:100).
(lanes 2 and 5), and RBL (lanes 3 and 6) cell lysates
A. Neuropeptide Y1 Receptor (red) appears in the striatal matrix
and rat brain lysates (lanes 7 and 8):
(asterisk) and in medium-size cells in the matrix (arrows).
1, 2, 3, 7. Anti-Neuropeptide Y1 Receptor antibody
B. Parvalbumin (green) appears in the striatal matrix.
(#ANR-021), (1:200).
C. confocal merge of Neuropeptide Y1 Receptor and parvalbumin
4, 5, 6, 8. Anti-Neuropeptide Y1 Receptor antibody
indicates that Neuropeptide Y1 Receptor is restricted to granule
preincubated with the control peptide antigen.
cells. DAPI is used as the counterstain (blue).
GPCR Pathways No.3 Winter 2010 www.alomone.com
39
Name
Cat. #
Indirect flow cytometry analysis of Neurotensin
Receptor 1 in live intact human HL-60 cells
Expression of Neurotensin Receptor 1 in breast
cancer
Neurotensin Receptors
Anti-Neurotensin Receptor 1
(extracellular)
ANT-015
Anti-Neurotensin Receptor 2
ANT-016
Anti-Neurotensin Receptor 1
(extracellular)
Cat. #: ANT-015
HL-60 cells + goat-anti-rabbit-FITC.
HL-60 cells + Anti-Neurotensin Receptor 1
Sizes: 50 µl | 0.2 ml
(extracellular) antibody (#ANT-015), (1:20) + goat-antirabbit-FITC.
Immunohistochemical staining of human breast cancer
sections. Staining (brown color) is specific for epitheliumderived malignant cells. Following EDTA pH 8 treatment,
slides were incubated overnight at 4°C with Anti-Neurotensin
Receptor 1 (extracellular) antibody (#ANT-015), (1:100).
Host: Rabbit.
Hematoxilin is used as the counterstain.
Epitope: Peptide EQNRSADGQHAGGLVC,
corresponding to amino acid residues
209-224 of human Neurotensin Receptor 1
(Accession P30989).
1
Applications: WB, IH, IC, IFC
2 3
4
5 6
150
100
75
Western blot analysis of human colon cancer HT-29 (lanes 1 and 4) human
50
adenocarcinoma MDA-MB-468 (lanes 3 and 6) cell lysates:
37
1-3. Anti-Neurotensin Receptor 1 (extracellular) antibody (#ANT-015), (1:200).
25
Anti-Neurotensin Receptor 2
Cat. #: ANT-016
lung small cell carcinoma NCI-H526 (lanes 2 and 5) and human breast
4-6. Anti-Neurotensin Receptor 1 (extracellular) antibody, preincubated with
the control peptide antigen.
Expression of Neurotensin Receptor 2 in rat
C6 cells
A
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide CGEQHSLVPLPQEAPE,
Expression of Neurotensin Receptor 2 in rat
stomach
corresponding to amino acid residues
377-392 of rat Neurotensin Receptor 2
(Accession Q63384 ).
Applications: WB, IH, IC
B
1
2
250
150
100
75
50
37
A. Immunocytochemical staining of paraformaldehydefixed and permeabilized rat C6 glioma cells with Anti-
Immunohistochemical staining of rat stomach sections.
Western blot analysis of mouse brain lysates:
Neurotensin Receptor 2 antibody (#ANT-016), (1:200),
Paraffin-embedded rat stomach sections were stained with
1. Anti-Neurotensin Receptor 2 antibody
followed by goat-anti-rabbit-AlexaFluor-555 secondary
Anti-Neurotensin Receptor 2 antibody (#ANT-016), (1:50),
(#ANT-016), (1:400).
antibody (orange staining). Nuclei were visualized with
(brown staining). Staining is specific for parietal cells
2. Anti-Neurotensin Receptor 2 antibody,
the cell permeable dye Hoechst 33342 (blue staining).
(arrows) of the gastric glands. Hematoxilin is used as the
preincubated with the control peptide antigen.
B. Live view of the field shown in (A).
counterstain.
40
GPCR Pathways No.3 Winter 2010 www.alomone.com
Name
Cat. #
Opioid Receptors
Anti-δ-Opioid Receptor (extracellular)
AOR-014
Anti-µ-Opioid Receptor (extracellular)
AOR-011
Anti-δ-Opioid Receptor (extracellular)
Cat. #: AOR-014
Expression of δ-Opioid Receptor in rat spinal cord
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide (C)ELVPSARAELQSSPLVN,
corresponding to amino acid residues 2-18 of mouse
δ-Opioid Receptor (Accession P32300).
Applications: WB, IH
Western blot analysis of rat cortex
lysate:
1. Anti-δ-Opioid Receptor (extracellular)
antibody, (#AOR-014), (1:200).
Immunohistochemical staining of rat spinal cord frozen sections using Anti-δ-Opioid
2. Anti-δ-Opioid Receptor (extracellular)
Receptor (extracellular) antibody (#AOR-014), (1:100), followed by goat-anti-rabbit
antibody, preincubated with the control
AlexaFluor-488 secondary antibody (green). Staining is present in neuronal cell
peptide antigen.
bodies (arrows). Hoechst 33342 (blue) is used as the counterstain.
Anti-μ-Opioid Receptor (extracellular)
Expression of µ-Opioid Receptor in rat spinal cord
Cat. #: AOR-011
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide CSPAPGSWLNLSHVDGN,
corresponding to amino acid residues 22-38 of rat
μ-Opioid Receptor (Accession P33535 ).
Applications: WB, IH
Western blot analysis of rat
hippocampus lysate:
1. Anti-μ-Opioid Receptor (extracellular)
Immunohistochemical staining of rat spinal cord frozen sections using Anti-µ-Opioid
antibody, (#AOR-011), (1:200).
Receptor (extracellular) antibody (#AOR-011), (1:100), followed by goat-anti-rabbit
2. Anti-μ-Opioid Receptor (extracellular)
AlexaFluor-488 secondary antibody (green). Staining is present in both neuronal cell
antibody, preincubated with the control
bodies (white arrows) and their prolongations (red arrows). Hoechst 33342 (blue) is
peptide antigen.
used as the counterstain.
GPCR Pathways No.3 Winter 2010 www.alomone.com
41
Name
Cat. #
Anti-Orexin Receptor 2
Cat. #: AOR-002
Orexin Receptors
Anti-Orexin Receptor 1
AOR-001
Sizes: 50 µl | 0.2 ml
Anti-Orexin Receptor 2
AOR-002
Host: Rabbit.
Epitope: Peptide (C)DRLARGRTSTESRKS,
corresponding to amino acid residues 391-405
of rat Orexin Receptor 2 (Accession P56719).
Applications: WB, IH, IC
Anti-Orexin Receptor 1
Cat. #: AOR-001
Expression of Orexin Receptor 2 in rat brain
A
Sizes: 50 µl | 0.2 ml
B
C
GL
Host: Rabbit.
ML
Epitope: Peptide (C)RNWKRPSEQLEAQH,
corresponding to amino acid residues 256-269
of rat Orexin Receptor 1 (Accession P56718).
Applications: WB, IH, IC
Immunohistochemical staining of rat brain sections using Anti-Orexin Receptor 2 antibody (#AOR-002), (1:50).
A. In the cerebellum, staining is present in cell bodies of both Golgi type I (Purkinje cells – white arrows) and Golgi type II
(green arrows) neurons. Staining is also present in fibers in the molecular layer (ML).
B. In the parietal cortex, staining is evident in neural cell bodies.
C. In the hippocampus, staining is present in pyramidal cells in the CA1 layer. Note that both cell bodies and prolongations
are stained.
Expression of Orexin Receptor 1 in rat colon
Following citrate treatment for antigen retrieval, slides were incubated overnight at 4°C with Anti-Orexin Receptor 2
antibody (1:50), followed by goat-anti-rabbit-AlexaFluor-555 secondary antibody (1:500), (red staining). Hoechst 33342
(blue) is used as the counterstain.
Expression of Orexin Receptor 2 in human colon cancer cells
M
A
SM
B
C
ME
Immunohistochemical staining of paraffin-embedded
longitudinal sections of rat colon showing mucosa (M),
submucosa (SM), and muscularis externa (ME). Staining
(red-brown color) is highly specific for absorptive cells in
the superior third of the intestinal glands. Following citrate
A. Immunocytochemical staining of paraformaldehyde-fixed and permeabilized human Colo-205 colon cancer cells with
treatment for antigen retrieval, slides were incubated
Anti-Orexin Receptor 2 antibody (#AOR-002), (1:500), followed by goat anti-rabbit-AlexaFluor-555 secondary antibody
overnight at 4°C with Anti-Orexin Receptor 1 antibody
(red staining).
(#AOR-001), (1:100). Immunolabeling was detected using
B. Live view of the field shown in (A).
DAB as the chromogen and hematoxilin as the counterstain.
C. Nuclei were visualized with the cell permeable dye Hoechst 33342 (blue staining).
Western blot analysis of rat brain
42
lysate:
Western blot analysis of rat (lanes 1 and 3) and
1. Anti-Orexin Receptor 1
mouse (lanes 2 and 4) brain lysates:
antibody (#AOR-001), (1:500).
1, 2. Anti-Orexin Receptor 2 antibody (#AOR-002),
2. Anti-Orexin Receptor 1
(1:400).
antibody, preincubated with the
3, 4. Anti-Orexin Receptor 2 antibody, preincubated
control peptide antigen.
with the control peptide antigen.
GPCR Pathways No.3 Winter 2010 www.alomone.com
Name
Cat. #
Anti-Prokineticin Receptor-1 (extracellular)
Cat. #: APR-041
Prokineticin Receptors
Anti-Prokineticin Receptor-1
(extracellular)
APR-041
Anti-Prokineticin Receptor-2
(extracellular)
APR-042
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide (C)ENTTNTFTDFFSARD, corresponding to
amino acid residues 10-24 of rat Prokineticin Receptor-1
(Accession Q8R416).
Applications: WB, IH, IFC
Anti-Prokineticin Receptor-2
(extracellular)
Expression of Prokineticin Receptor-1 in mouse olfactory bulb brain sections
B
A
Cat. #: APR-042
G
G
G
G
G
G
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide (C)DQNGNTSFAPDLN,
corresponding to amino acid residues 3-15 of rat
Prokineticin Receptor-2 (Accession Q8R415).
Applications: WB, IH
Immunohistochemical staining of mouse olfactory bulb sections with Anti-Prokineticin
Expression of Prokineticin Receptor-2 in rat DRG
Receptor-1 (extracellular) antibody (#APR-041), (1:100).
A. Prokineticin Receptor-1 reveals intensely stained astrocyte-like cells in the glomeruli
(G) and lightly stained astrocyte-like cells in the adjacent layer (arrows, green
fluorescence).
B. Merge of images of Prokineticin Receptor-1 (green), parvalbumin (red) and DAPI (blue)
nuclear counterstain.
Expressin of Prokineticin Receptor-1 in rat DRG
Immunohistochemical staining of rat dorsal root ganglions
(DRG) frozen sections with Anti-Prokineticin Receptor-2
(extracellular) antibody (#APR-042), (1:100), followed by
goat-anti-rabbit AlexaFluor-594 secondary antibody (red
Immunohistochemical staining of frozen sections
staining). Staining is present in small, medium and large DRG
of rat dorsal root ganglion (DRG) with Anti-
neurons. Hoechst 33342 (blue) is used as the counterstain.
Prokineticin Receptor-1 (extracellular) antibody
(#APR-041), (1:100), followed by goat-anti-rabbitAlexaFluor-555 secondary antibody. Prokineticin
Receptor 1 (red staining) is expressed in DRG
1
neurons. Cell nuclei are visualized with Hoechst
2
33342 (blue staining).
130
95
72
55
1
33
Western blot analysis of rat brain membranes:
1. Anti-Prokineticin Receptor-2 (extracellular) antibody
(#APR-042), (1:200).
2. Anti-Prokineticin Receptor-2 (extracellular) antibody,
preincubated with the control peptide antigen.
GPCR Pathways No.3 Winter 2010 www.alomone.com
2
130
95
72
Western blot analysis of mouse brain lysate:
55
1. Anti-Prokineticin Receptor-1 (extracellular) antibody
36
28
(#APR-041), (1:200).
2. Anti-Prokineticin Receptor-1 (extracellular) antibody,
preincubated with the control peptide antigen.
43
Name
Cat. #
Proteinase-activated Receptors
Anti-Human Proteinase-activated
Receptor-1 (extracellular)
APR-031
Anti-Proteinase-activated Receptor-2
APR-032
Anti-Proteinase-activated Receptor-4
(extracellular)
APR-034
Anti-Proteinase-activated Receptor-4
(extracellular)-FITC
APR-034-F
Expression of Proteinase-activated
Receptor-1 in normal human breast
and human breast carcinoma
Anti-Proteinase-activated Receptor-4
(extracellular)
Cat. #: APR-034
A
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Epitope: Peptide (C)HLRGQRWPFGEAA(S)R,
corresponding to amino acid residues 136-150 of
human Proteinase-activated Receptor-4 (Accession
Q96RI0). Cys 149 was replaced with Ser.
Applications: WB, IFC, IC, IH
B
Expression of Proteinase-activated Receptor-4
in rat epididymis
Anti-Human Proteinase-activated
Receptor-1 (extracellular)
Cat. #: APR-031
Immunohistochemical staining of paraffin-embedded
section showing normal resting breast (A) and breast
Sizes: 50 µl | 0.2 ml
carcinoma (B). Proteinase-activated Receptor-1 staining is
Host: Rabbit.
Epitope: Peptide (C)KNESGLTEYRLVSINK,
corresponding to amino acid residues 61-76
of human Proteinase-activated Receptor-1
highly specific for epithelium-derived cells.
Following citrate treatment, slides were incubated overnight
at 4°C with Anti-Human Proteinase-activated Receptor-1
(extracellular) antibody (#APR-031), (1:100). Color reaction
(Accession P25116 ).
was obtained using Histofine (pink) (A) or DAB (brown) (B).
Applications: WB, IFC, IC, IH
Hematoxilin was used as the counterstain in both cases.
Immunohistochemical staining of rat epididymis paraffin-
Anti-Proteinase-activated Receptor-2
embedded sections using Anti-Proteinase-activated
Cat. #: APR-032
Note that strong and specific staining is present in both
Receptor-4 (extracellular) antibody (APR-034), (1:100).
the pseudostratified epithelium (blue arrow) and the
smooth muscle cells of the muscular blood vessels (red
Sizes: 50 µl | 0.2 ml
arrow). Hematoxilin is used as the counterstain.
Host: Rabbit.
Epitope: Peptide (C)KRMQISLTSNKFSRK,
corresponding to amino acid residues 368-
Western blot analysis of human breast adenocarcinoma
MDA-MB-231 (lanes 1 and 4) and MDA-MB-468 (lanes 2
382 of rat Proteinase-activated Receptor-2
and 5), and human lung small cell carcinoma NCI-H526
(Accession Q63645).
(lanes 3 and 6) cell lines:
Applications: WB, IC
1-3. Anti-Proteinase-activated Receptor-2 antibody (#APR032), (1:600).
4-6. Anti-Proteinase-activated Receptor-2 antibody,
preincubated with the control peptide antigen.
Indirect flow cytometry analysis of Proteinaseactivated Receptor-4 in human HL-60 cells
Expression of Proteinase-activated Receptor-2 in human PC3 cells
A
B
C
A) Immunocytochemical staining of paraformaldehyde-fixed and permeabilized human prostate carcinoma PC3 cells with AntiProteinase-activated Receptor-2 antibody (#APR-032), (1:500), followed by goat-anti-rabbit-AlexaFluor-555 secondary antibody
(orange staining).
HL-60 cells + goat-anti-rabbit-FITC.
HL-60 cells + Anti-Proteinase-activated Receptor-4
B) Nuclei were visualized with the cell-permeable dye Hoechst 33342 (blue staining)
(extracellular) antibody (#APR-034), (1:40) + goat-anti-
C) Merge of A and B.
rabbit-FITC.
44
GPCR Pathways No.3 Winter 2010 www.alomone.com
Anti-Proteinase-activated Receptor-4
Name
(extracellular)-FITC
Cat. #
Name
Somatostatin Receptors
Cat. #: APR-034-F
Anti-Somatostatin Receptor Type 5
(extracellular)
Cat. #
Sphingosine-1-Phosphate Receptors
Anti-Sphingosine 1-Phosphate
Receptor 1 (extracellular)
ASR-005
ASR-011
Sizes: 50 µl
Host: Rabbit.
Epitope: Peptide (C)HLRGQRWPFGEAA(S)R,
corresponding to amino acid residues 136-150 of
human Proteinase-activated Receptor-4 (Accession
Q96RI0 ). Cys 149 was replaced with Ser.
Applications: FC
Flow cytometry analysis of Proteinase-activated
Receptor-4 in live intact HL-60 cells
Anti-Somatostatin Receptor Type 5
Anti-Sphingosine 1-Phosphate Receptor 1
(extracellular)
(extracellular)
Cat. #: ASR-005
Cat. #: ASR-011
Sizes: 50 µl | 0.2 ml
Sizes: 50 µl | 0.2 ml
Host: Rabbit.
Host: Rabbit.
Epitope: Peptide CEPLSLASTPSWNAS, corresponding
Epitope: Peptide SDYVNYDIIVRHYN(C) corresponding
to residues 2-15 of rat Somatostatin Receptor Type 5
to amino acid residues 17-30 of human Sphingosine-1-
(accession P30938).
Phosphate Receptor 1 (Accession P21453).
Applications: WB, IH
Applications: WB, IH, IC
Expression of Somatostatin Receptor Type 5
in mouse cerebellum
A
B
Expression of Sphingosine-1-Phosphate
Receptor 1 in rat lung
C
Unstained HL-60 cells.
HL-60 cells + Anti-Proteinase-activated Receptor-4
(extracellular)-FITC antibody (#APR-034-F), (1:40).
Immunohistochemical staining of paraffin embedded
Flow cytometry analysis of Proteinase-activated
Receptor-4 expression in human HL-60 cells
rat lung using Anti-Sphingosine 1-Phosphate Receptor 1
(extracellular) antibody (#ASR-011), (1:100) followed by
SuperPicture HRP-conjugated polymer (Zymed). Staining is
present in vascular smooth muscle (black arrows) but not
in the muscular layer of bronchi (red arrows). Hematoxilin is
used as the counterstain.
Immunohistochemical staining mouse cerebellum using
Anti-Somatostatin Receptor Type 5 (extracellular) antibody
(#ASR-005), (1:100).
A. Somatostatin Receptor Type 5 (red) appears in Purkinje
cells (horizontal arrows) and in molecular layer interneurons
(vertical arrows). B. Parvalbumin (green) appears in the
Purkinje cells and their processes.
C. Merge of Somatostatin Receptor Type 5 and parvalbumin
demonstrates co-localization in Purkinje cells (horizontal
arrows) but not in the molecular layer (vertical cells). The
blue counterstain is DAPI.
1
2
3 4
250
130
95
72
55
36
28
17
Western blot analysis of Sphingosine-1-Phosphate
HL-60 cells were induced to differentiate into a mature
neutrophil-like phenotype by incubation with dimethyl
Western blot analysis of rat brain lysates:
Receptor 1 in RAEC cells (lanes 1 and 3) and in A-10
sulfoxide (DMSO) (1.25%) for three days. The neutrophil
1. Anti-Somatostatin Receptor Type 5
(lane 2 and 4) cell lysates:
phenotype was confirmed by staining the cells with Anti-
(extracellular) antibody (#ASR-005),
1, 2. Anti- Sphingosine-1-Phosphate Receptor 1
CD11b-PE antibody. Double staining with Anti-Proteinase-
(1:200).
(extracellular) antibody (#ASR-011), (1:200).
activated Receptor-4 (extracellular)-FITC antibody (#APR-
2. Anti-Somatostatin Receptor Type 5
3, 4. Anti- Sphingosine-1-Phosphate Receptor 1
034-F), (1:40) shows that most differentiated cells express
(extracellular) antibody, preincubated
(extracellular) antibody, preincubated with the
both markers (upper right panel).
with the control peptide antigen.
control peptide antigen.
GPCR Pathways No.3 Winter 2010 www.alomone.com
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