Western Blot Analysis of SNARE Proteins

Cells were directly lysed with Laemmli sample buffer supplemented with protease inhibitors (Roche). Cell lysates were loaded onto NuPage 4–12% Bis-Tris gels (Life Technologies) and separated by electrophoresis in MOPS buffer. Proteins were transferred onto Protran nitrocellulose membranes (Whatman) and saturated for 1 h in PBS-T (PBS, 0.1% Tween 20) supplemented with 5% non-fatty milk. anti-SNAP25 (SMI-81, 1:10,000) was from Biologend; anti-VAMP-2 (104211, 1:2000) was from Synaptic System; anti-SNAP25 BoNT/A-cleaved (1:5000) was home made and used as previously described^{71 (link)}; anti-Syntaxin-1A/1B (1:2000) was home made and used as previously described^{72 (link)}. Incubation with indicated primary antibodies was performed overnight at 4 °C. The membranes were then washed three times with PBS-T and incubated with appropriate HRP-conjugated secondary antibodies (Anti mouse 1:5000 and Anti rabbit& mouse Flourescent labeled 1:2000) for 1 h. Membranes were washed three times with PBS and proteins revealed with an Uvitec gel doc system (Uvitec Cambridge).