VAMP and SNAP-25 are present in platelets and interact with syntaxin 4. (A) Detection of VAMP and SNAP-25 in platelet membranes. Platelet membranes (50 μg) solubilized in Triton X-100 (1%) were subjected to SDS-PAGE on 15% gels. Proteins were electroblotted to polyvinylidene membranes. The membranes were probed with monoclonal anti-VAMP antibody (SP10, 1:500 dilution) or monoclonal anti-SNAP-25 antibody (SP12, 1 μg/mL). Bound antibody was detected by an enhanced chemiluminescence method with a rabbit antimouse antibody coupled to peroxidase. Relative positions of molecular mass standards are indicated in kilodaltons. (B) VAMP and SNAP-25 coimmunoprecipitate with syntaxin 4. Triton X-100 (1%) solubilized platelet membranes (400 μL) were incubated overnight at 4°C with affinity-purified syntaxin 4 antibody or a control antibody. After washing, the bound proteins were eluted with sample buffer and subjected to SDS-PAGE and to immunoblotting with the anti-VAMP or anti-SNAP-25 antibodies as just described. Relative positions of molecular mass standards are indicated in kilodaltons.