α synaptophysin

Antibodies against (α-) Piccolo (rabbit), Bassoon (mouse), and MAP2 (rabbit and mouse) were used as previously described [41 (link)]. α-Tubulin (mouse) antibodies were from Sigma, and α-PSD-95 (mouse) was from Affinity BioReagents. The following antibodies were purchased from Santa Cruz: α-Synaptophysin (rabbit), α-Trio (C-terminal antibody; goat), and α-Myc (rabbit). The mouse Trio α-GEF2 antibody was from Abnova. Mouse α-Synaptotagmin was purchased from BD Biosciences. The rabbit α-GFP antibody was from Invitrogen. The α-ELKS2 antibody was generated in rabbits using a commercial vendor (Washington Biotechnology). The epitope was E. coli purified GST-tagged amino acids 107–138 of ELKS2 [same region as used by [16 (link)]]. The serum was passed over a column of GST coupled to Actigel ALD using manufacturer’s protocol (Sterogene Bioseparations) to remove antibodies directed against GST. Antibody was then affinity purified with the antigen coupled to Actigel ALD.

Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in RIPA buffer (50 mM Tris.HCl, pH 7.4, 150 mM NaCl, 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate (SDS), 2 mM PMSF, and 1× protease inhibitor cocktail (Roche, Indianapolis, IN)) on ice for 20 min for whole cell lysate preparation. Protein concentration was determined using a Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA), and the optical density at 650 nm was taken using a Bio-Rad iMark microplate reader (Bio-Rad). Cell lysates were electrophoretically separated by 8–12 % sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels and Western blot analysis with the following primary antibodies: α-CREB, α-PCREB, α-BDNF (for the mature form of BDNF), α-p250GAP, α-MecP2, α-synaptophysin, α-PSD-95, α-C-MYC, α-CBP (Santa Cruz Biotechnology, Dallas, TX), α-β-actin (Sigma, St. Louis, MO), α-MAP-2 (EMD Millipore, Billerica, MA), α-His (Applied Biological Materials ABM, Richmond, BC), and α-p24 (NIH AIDS Reagents Program, donated by Dr. Bruce Chesebro of NIAID) [73 (link)]. Sheep anti-mouse IgG-HRP and donkey anti-rabbit IgG-HRP from GE Healthcare (Marlborough, MA) were used as secondary antibodies, followed by ECL detection and imaging using a Bio-Rad ChemiDoc imaging system (Bio-Rad).