Figure 6.

Infected wild-type and 12-insertion mutant of synaptobrevin 2 engage in assembled SNARE complexes. A, Cells from each six-well plate were harvested using 1 ml of buffer, and proteins were extracted using 1% Triton X-100. A total of 50 μl of cell lysate was used for SDS-PAGE and Naphto blue black staining. B, A total of 10 μl of rabbit serum raised against rat syntaxin 1 (U6251) and 20 μl of protein A-Sepharose beads were added to 1% Triton X-100 cell lysate expressing mutant synaptobrevin 2 to immunoprecipitate (IP) assembled SNARE complexes. After incubation, beads were washed five times, and proteins coimmunoprecipitated with syntaxin 1 were determined by Western blotting. Shown are Western blots of proteins coimmunoprecipitated with syntaxin 1. Blots were performed using the following sera or ascites: mouse anti-GFP, C163 from Zymed; mouse anti-Syb 2, CL69.1; mouse anti-syntaxin 1, HPC-1; and mouse anti-SNAP-25, CL71.1.