Rabbit anti synapsini

Cells were collected with a cell scraper in ice-cold PBS (pH 7.4) and centrifuged at 1000 rpm for 5 min in a precooled centrifuge at 4°C. The cell pellet was resuspended in ice-cold lysis buffer (20 mM Tris-HCl, pH 8, 150 mM KCl, 1% Triton X-100, 1 mM PMSF and Complete protease inhibitor cocktail (Roche)), incubated on ice for 10 min, followed by centrifugation at 13,200 rpm for 10 min at 4°C. The supernatant was collected, NuPAGE LDS sample buffer (Invitrogen) containing 2% β-mercaptoethanol was added and samples were boiled for 5 min at 90°C. Proteins were separated in 8% SDS-PAGE gels and transferred onto nitrocellulose membrane (Hybond-C Extra; Amersham). Membranes were incubated in blocking buffer [PBS, 0.05% (v/v) Tween 20 and 5% milk powder] for 30 min at RT. Membranes were incubated with corresponding primary antibodies in blocking buffer overnight at 4°C. Antibodies used: rat anti-GFP (Chromotek) 1:1000; rat anti-HA (Roche) 1:100, rabbit anti-Cntn6-45 (Harlan) 1:2000; chick anti-Lphn1-p120 and rabbit anti-Lphn1-p85 1:2500, rabbit anti-SynapsinI (Sigma) 1:2000, mouse anti-PSD95 (Milipore) 1:250, mouse anti-βActin (Sigma) 1:1000. Blots were incubated with SuperSignal West Dura Extended Duration Substrate (Pierce) and exposed to ECL films (Pierce) or imaged by FluorchemE Digital Darkroom (Cell Biosciences). ImageJ was used for blot quantification.