Living colors anti gfp

The following primary antibodies were from Cell Signaling: Anti-WAVE3, anti-YB1, anti-Lamin-B1 and anti-α-Tubulin. Anti-β-Actin was from Sigma, while living colors anti-GFP and anti-mouse GFP were from Clontech and Santa Cruz Biotechnology Inc., respectively. All primary antibodies were used in 1:1,000 dilution. Goat horseradish peroxidase-conjugated anti-mouse IgG (1:2,000) and goat horseradish peroxidase-conjugated anti-rabbit IgG (1:2,000) were from Calbiochem. Vecta-shield with 4′,6-diamidino-2-phenylindole was from Vector Laboratories. All gel electrophoresis reagents were from BioRad.

Hippocampal neurons from Munc13-1/2 DKO expressing Munc13–1 full length, Munc13–1 V549E,L554E, Munc13–1 R750E,K752E and Munc13–1 N940W mutants were lysed after 15 DIV at 4°C with RIPA lysis buffer including protease inhibitor cocktail-complete mini (Roche Diagnostics, Berlin, Germany). Equal amounts of proteins from the lysates of the four different groups were mixed with Laemmli sample buffer containing 0.1 M DTT, and boiled 5 min at 99°C. Protein lysates were separated on SDS polyacrylamide gels (4–8%% SDS-PAGE) and transferred to a polyvinyl difluoride (PVDF) membrane. Membranes were blocked for 1 hr with 5% skim milk in TBST and incubated at 4°C over night with primary antibodies: anti-Flag M2 (F1804; Sigma-Aldrich), and anti-Living Colors GFP (632375; Clontech, Mountain View, CA). Secondary antibodies were horseradish peroxidase-conjugated (Jackson ImmunoResearch, West Grove, PA). The immunoreactive proteins were detected by ECL Plus Western Blotting Detection Reagents (GE Healthcare Biosciences, Pittsuburgh, PA) in a Fusion FX7 detection system (Vilber Lourmat, Eberhardzell, Germany). Data were collected from three separate Munc13-1/2 DKO cultures and analyzed offline using ImageJ.