The proteins were then diluted with 2X SDS loading buffer [4% SDS, 125 mM (pH 6.8) tris-HCl, 20% glycerol, 5% 2-mercaptoethanol, and 0.01% bromophenol blue] and denatured by heating to 95°C for 5 min. After cooling, the denatured protein samples were loaded into separate wells of 10% TGX gels (Bio-Rad, catalog #4561033 DC) along with a precision plus protein (10 to 250 kDa) dual color standards (Bio-Rad, catalog #1610374) ladder. Western blots were run for 2 hours at 80 volts and 400 mA. Proteins from the gel were transferred to a 0.45-μm nitrocellulose membrane (Bio-Rad, catalog #1620115) using the Bio-Rad transferring system for 2 hours at a constant 100 V at 4°C. The nitrocellulose membrane was blocked in 5% nonfat milk for 1 hour at room temperature and then incubated with primary antibodies (1:1000; synapsin 1, Synaptic Systems, RRID: AB_2619772; synaptotagmin 1, Santa Cruz Biotechnology, catalog #sc-136480; PSD95, Synaptic systems, catalog #124011; syntaxin, I378, custom made, gift of T. Südhof) overnight at 4°C. The following day, the blots were washed three times with tris-buffered saline with 0.1% Tween 20 (TBST)
