Proteins were analyzed by Western blot analysis with monoclonal
antibodies to 12 known synaptic proteins. The 12 proteins examined were
SNAP-25, syntaxin-4, syntaxin-6, rsec8, rabaptin-5, munc-18,
complexin-2, synaptotagmin, synapsin IIA, rabphilin3a, doc2, and
synaptogyrin (Synaptic Vesicle Sampler Kit, Transduction Laboratories,
Lexington, KY). Fifty micrograms of each retinal protein sample and
prestained standards (Kaleidoscope; Bio-Rad, Hercules, CA) was
electrophoresed in a 12% (wt/vol) sodium dodecyl
sulfate-polyacrylamide gel and transferred to polyvinylidene difluoride
membrane (PVDF; Immobilon-P; Millipore, Bedford, MA). Nonspecific
protein binding sites were blocked in a solution containing 5% dry
milk and 0.1% Tween 20 in Tris-buffered saline (TBS; 20 mM Tris-HCl[
pH7.6] and 137 mM NaCl) for 2 hours.
Membranes were then incubated with the primary antibodies at dilutions
of 1:250 to 1:5000 in the blocking solution for 2 hours. After three
washes in TBS-T (0.1% Tween 20 in TBS), the membranes were incubated
with the secondary antibody (peroxidase-labeled anti-mouse IgG
antibody; Amersham Pharmacia Biotech, Piscataway, NJ) at a dilution of
1:1000 in TBS-T for 1 hour. Finally, the membranes were washed five
times in TBS-T, and detection was performed by enhanced
chemiluminescence (ECL; Amersham Pharmacia Biotech). Rat brain lysate
was used as the positive control protein for the detection of synapsin
IIa, munc-18, rabphilin-3A, SNAP-25, synaptotagmin, and complexin-2;
rat pituitary lysate for synaptogyrin and syntaxin-6; human endothelial
lysate for doc2 and syntaxin-4; MDCK lysate for rsec8; and MCF-7 lysate
for rabaptin-5. All positive control protein lysates were provided by
Transduction Laboratories. All the blots were reacted simultaneously
with each antibody and processed identically, and the analysis was
performed two to three times.