Download supplement

Supporting Online Material:
Comment on "Nervy Links Protein Kinase A to Plexin-Mediated Semaphorin
Repulsion" by Ice et al.
Materials and Methods
Comparison of Drosophila embryos stained with anti-ETO Ab-1 and anti-Nvy
(Fig. 2 in main text). Overnight collections of nvyPDFKG1/CyO, Kr>GFP or Oregon-R
embryos were fixed and prepared for confocal microscopy as previously described (1).
Rabbit anti-ETO was used at the same concentration as previously described (1); Rabbit
anti-Nvy antisera was generated against GST-tagged, Nvy expressed and purified from E.
coli (see Supplemental methods). Pre-absorbed rabbit anti-Nvy was used at 1:300; rat
anti-Elav 7E8A10 (Hybridoma Bank) was used at 1:50; and mouse anti-FasII 1D4
(Hybridoma Bank) was used at 1:10.
Anti-Nvy antibody. Rabbit anti-Nvy polyclonal antibody was generated against a
GST-Nvy fusion protein purified after expression in E. coli. The fragment of Drosophila
Nvy that was fused to GST was from amino acid 178 to 743 (which includes all of the
domains showing similarity to ETO) and cloned into the pGEX2T vector (Amersham
Biosciences). Expression and purification of GST-Nvy were carried out as recommended
by Amersham Biosciences. PAGE gel slices containing Coomassie-stained Nvy protein
were shipped to Cocalico Biologicals (Reamstown, PA) for injections into rabbits. The
resulting antisera showed excellent immunoreactivity in Drosophila tissues after the third
boost.
Anti-Nvy immunoprecipitation. 100 µL of AG11-Gal4 UAS-nvy embryos were
homogenized in 500 µL RIPA buffer (50 mM Tris pH 7.5, 150mM NaCl, 20 mM
MgCl2, 0.5% NP-40, 0.5 mM PMSF + protease inhibitors). 1 µL of anti-Nvy antisera or
rabbit pre-immune serum was incubated with 125 µL of embryonic lysate for 8 hr at 4˚C.
Next, the reactions were incubated with 20 µL agarose beads for 1 hr. The beads were
spun down, washed 3X 10 min, and re-suspended in 20 µL RIPA+SDS loading buffer.
The samples were separated in an SDS-PAGE gel, which was probed with anti-Nvy
(1/850).
Fly stocks and immunofluorescence. UAS-nvy-RNAi is a cDNA/genomic DNA
hybrid created using the guidelines described in (2, 3). Nucleotides 1314-1889 of the nvy
cDNA were fused to the complimentary nvy genomic region and cloned into pUASt.
Mouse anti-Pros and anti-Elav, from the Developmental Studies Hybridoma Bank, were
used at 1:2 and 1:50, respectively. Preabsorbed rabbit anti-Nvy was used at a final
dilution of 1:300.
Immunoblot analysis. Plasmids expressing the indicated proteins from the CMV
immediate early promoter were transfected into Cos7 cells and cellular extracts analyzed
by immunoblot analysis using the indicated antisera after separation of proteins by 10%
SDS polyacrylamide gel electrophoresis. Antibody dilutions ranged from 1:200 to 1:2000
with similar results.
Supporting References:
1. J. Hirata, H. Nakagoshi, Y. Nabeshima, F. Matsuzaki, Nature 377, 627 (Oct 19, 1995).
2. S. Kalidas, D. P. Smith, Neuron 33, 177 (Jan 17, 2002).
3. E. P. Spana, C. Q. Doe, Development 121, 3187 (Oct 1995).
Fig. S1. Neither lot of Anti-ETO Ab-1 recognizes nervy, even upon long exposures.
Extracts of Cos7 cells expressing Gal4-tagged forms of MTG8/ETO, MTG16, and Mtgr1
(the mammalian homologs of Nvy), a dual epitope tagged (Gal4 and Myc) form of Nervy
(Gal4-Nvy), and Myc-tagged Nervy (Myc-Nvy) were analyzed by immunoblot using
both lots of serum (A and B) that have been supplied to EMD Biosciences. The *
indicates background bands that appeared when the blots were over exposed.
Fig. S2. Additional characterization of the anti-Nvy antibody and Nvy staining
pattern during Drosophila development. All of the Nvy immunostains, western blots,
and immunoprecipitations were carried out with the same rabbit anti-Nvy antibody,
preabsorbed overnight against wild type Drosophila embryos. (A) Larval eye imaginal
disc with clones expressing two copies of nvy-RNAi (yw122; UAS-nvy-RNAi/+;
tubulin>GFP, y+>Gal-4/UAS-nvy-RNAi) and stained with the anti-Nvy antibody. The
RNAi-expressing clones, which are marked by the absence of GFP (green), have
undetectable levels of Nvy (red or white). The outline of the clone border is traced in the
right-hand panel (A’). The elimination of anti-Nvy immunoreactivity in the nvy RNAiexpressing clones strongly suggests that this antibody recognizes the product of the nvy
cDNA. (B) Anti-Nvy antisera immunoprecipitates Nvy from embryos. The immunoblot
was probed with anti-Nvy. Under these conditions, Nvy migrates slightly slower than its
predicted Mw of 76,500 Da, which is consistent with the migration of Myc-Nervy (Fig.
1). Lane 1: 12% of the extract used in the immunoprecipitations; lane 2: supernatant of
embryonic lysate that was incubated with anti-Nvy, showing that Nvy is
immunodepleted; lane 3: supernatant of embryonic lysate that was incubated with rabbit
pre-immune serum showing no depletion of Nvy; lane 4: anti-Nvy immunoprecipitate;
lane 5: rabbit pre-immune serum immunoprecipitate. (C) Ventral view of a stage 11
embryo stained with anti-Nvy (red) and anti-Prospero (green).
Prospero marks the
neuroblasts (NB) and their progeny, the ganglian mother cells (GMC). At this stage, Nvy
is detected in delaminating neuroblasts. This stage of embryogenesis is soon after Nvy
protein is robustly detected with this antibody. (D) Magnified view of the midline region
of the embryo shown in (C). Nvy (red) is localized to the nuclei of NBs and GMCs.
Pros (green) is cytoplasmic (arrow) in most NBs (except for the MP2 NB, in which it is
nuclear; note that the MP2 nucleus is also very large) and nuclear in the GMCs
(arrowhead) (1). (E). Blastoderm stage embryo stained with anti-Nvy. There is no
detectable Nvy staining, indicating a lack of maternal expression. (F). Lateral view of a
stage 13 embryo showing that Nvy (red) is detected in many PNS nuclei, which are also
marked by Elav (green).
GA
L
GA -ETO
GAL-MT /MTG
GAL-Mt G16 8
My L-Negr1
c-N rv
erv y
y
A. Anti-ETO
Ab-1
Lot #1
B. Anti-ETO
Ab-1
Lot #2
194
115
97
53
*
*
*
203
116
92
*
*
53
*
*
Supplemental Fig. 1
Supplemental Fig. 2