Figure 3.

Association of SNAP-29 with syntaxin-1 and SNARE complex. (A) Immunoprecipitation of syntaxin-1A with SNAP-29 from cotransfected HEK293 T cells. The syntaxin-1A–SNAP-29 complex was immunoprecipitated from cell extracts using anti-SNAP-29 antiserum (anti-29) followed by immunoblot with antibody to syntaxin-1. Normal rabbit IgG (IgG) was used as a control. (B) Immunoprecipitation of syntaxin-1 with SNAP-29 from solubilized rat brain homogenates. Syntaxin-1 was immunoprecipitated by anti-SNAP-29 antiserum and by anti-VAMP, but not by preimmune serum (preserum) or normal rabbit IgG (IgG). A 10% volume of brain homogenate (b.h.) was loaded (right lane). (C) Immobilized GST–SNAP-29 (GST-29) or GST alone on Sepharose beads was incubated with 100 μg of rat brain homogenates at 4°C for 3 h. Native SNARE complex captured was subjected to SDS/PAGE at temperatures as indicated and then analyzed by immunoblotting with the antibodies against syntaxin-1, SNAP-25, and VAMP-2. (Lower) Immunoblotting on the same membrane with anti-GST antibody.