Download Anti-Fibroblast Growth Factor Receptor 1 [pTyrpTyr653/654

RESEARCH USE ONLY
POLYCLONAL ANTIBODY
Anti-Fibroblast Growth Factor Receptor 1 [pTyrpTyr 653/654]
Phosphospecific Antibody, Unconjugated
Code No.
AT-2031
Isotype:
Rabbit IgG
Quantity:
100μL (10 mini-blot size)
BACKGROUND:
Fibroblast growth factor receptor 1 (FGFR1) is a member
of the fibroblast growth factor tyrosine kinase receptor
family (FGFR1, FGFR2, FGFR3, and FGFR4) and plays
diverse roles in various developmental and physiological
processes. FGFR1 is activated upon ligand binding to its
extracellular domain, resulting in its dimerization, and
activation of the kinase domain, leading to its
phosphorylation at specific tyrosine sites. FGFR1
contains at least seven tyrosine phosphorylation sites (Tyr
463, Tyr 583, Tyr 585, Tyr 653, Tyr 654, Tyr 730 and Tyr
766). Phosphorylation of tyrosines 653 and 654 in the
activation domain is critical for FGFR1 catalytic activity
and for mediating its biological responses.
PRODUCT:
Rabbit polyclonal immunoglobulin in Dulbecco’s
phosphate buffered saline (without Mg2+ and Ca2+), pH
7.3 (+/- 0.1), 50% glycerol, with 1.0 mg/mL BSA (IgG,
protease free) as a carrier. 0.05% sodium azide
IMMUNOGEN:
The antiserum was produced against a chemically
synthesized phosphopeptide derived from the region of
human FGFR that contains tyrosine 653 and 654. The
sequence is conserved in human, mouse and rat.
PURIFICATION:
Purified from rabbit serum by sequential epitope-specific
chromatography. The antibody has been negatively
preadsorbed using a non-phosphopeptide corresponding
to the site of phosphorylation to remove antibody that is
reactive with non-phosphorylated FGFR. The final
product is generated by affinity chromatography using a
FGFR-derived peptide that is phosphorylated at tyrosines
653 and 654.
SPECIFICITY:
Human, mouse and rat. This antibody may cross-react
with phosphorylated FGFR2 [pYpY656/657] and FGFR3
[pYpY647/648] as determined by peptide competition.
APPLICATIONS:
The antibody has been used in Western blotting and
immunofluorescent staining applications. For Western
blotting applications, we recommend using the antibody
at a 1:1000 dilution. For immunofluorecence staining
applications, we recommend a 1:50 dilution. The optimal
antibody concentration should be determined empirically
for each specific application.
15 B Constitution Way · Woburn, MA 01801 · Phone: 1.800.200.5459 · Fax: 781-939-6963 · www.mblintl.com
RESEARCH USE ONLY
STORAGE:
Store at −20oC. We recommend a brief centrifugation
before opening to settle vial contents. Then, apportion
into working aliquots and store at −20oC. For shipment or
short-term storage (up to one week), 2-8oC is sufficient.
POSITIVE CONTROL:
Thyroid carcinoma cells (TT cells), neuroblastoma cells
(Neuro 2A) cells, FGF2-treated 3T3 fibroblast cells, L6
myoblast cells.
REFERENCES:
St. Bernard, R., et al. (2005) Fibroblast growth factor
receptors as molecular targets in thyroid carcinoma.
Endocrinology 46(3):1145-1153.
Gowardhan, B., et al. (2005) Evaluation of the fibroblast
growth factor system as a potential target for therapy in
human prostate cancer. Br. J. Cancer 92(2):320-327.
Wang, F., et al. (2004) Chronic activity of ectopic type 1
fibroblast growth factor receptor tyrosine kinase in
prostate epithelium results in hyperplasia accompanied by
intraepithelial neoplasia. Prostate 58(1):1-12.
Hajihosseini, M.K., et al. (2004) Skeletal development is
regulated by fibroblast growth factor receptor 1 signalling
dynamics. Development 131(2):325-335.
Billottet, C., et al. (2004) Targets of fibroblast growth
factor 1 (FGF-1) and FGF-2 signaling involved in the
invasive and umorigenic behavior of carcinoma cells. Mol.
Biol. Cell 15(10):4725-4734.
Freeman, K.W., et al. (2003) Conditional activation of
fibroblast growth factor receptor (FGFR) 1, but not
FGFR2, in prostate cancer cells leads to increased
osteopontin induction, extracellular signal-regulated
kinase activation, and in vivo proliferation. Cancer Res.
63(19):6237-6243.
Stachowiak, M.K., et al. (1996) Nuclear accumulation of
fibroblast growth factor receptors is regulated by multiple
signals in adrenal medullary cells. Mol. Biol. Cell
7(8):1299-1317.
Campbell, J.S., et al. (1995) Differential activation of
mitogen-activated protein kinase in response to basic
fibroblast growthfactor in skeletal muscle cells. Proc.
Nat’l. Acad. Sci. USA 92(3):870-874.
15 B Constitution Way · Woburn, MA 01801 · Phone: 1.800.200.5459 · Fax: 781-939-6963 · www.mblintl.com
RESEARCH USE ONLY
Western Blotting Experiments
Lysates prepared from TT cells (lanes 1-5) were resolved by
SDS-PAGE on a 6% polyacrylamide gel and transferred to PVDF.
Membranes were left untreated (lanes 1-4) or treated with lambda
phosphatase (lane 5), blocked with a 3% BSA-TBST buffer for one
hour at room temperature, and incubated with the FGFR1 [pYpY
653/654
] antibody for two hours at room temperature in 3%
BSA-TBST buffer, following prior incubation with: no peptide
(lane 1), the non-phosphopeptide corresponding to the immunogen
(lane 2), a generic phosphotyrosine-containing peptide (lane 3), or
the phosphopeptide immunogen (lane 4). After washing,
membranes were incubated with goat F(ab’)2 anti-rabbit IgG HRP
conjugate and bands were detected using the Pierce SuperSignal™
method. The data show that only the peptide corresponding to
FGFR1 [pYpY653/654] blocks the signal and that phosphatase
stripping eliminates the signal, verifying that the antibody is site
phospho-specific.
15 B Constitution Way · Woburn, MA 01801 · Phone: 1.800.200.5459 · Fax: 781-939-6963 · www.mblintl.com