Platelet syntaxin 2 and syntaxin 4 participate in 20S complex formation. The 20S particle formation assay9 was used to show that syntaxin 2 and 4 behave as SNAREs in platelets. HPE, α-, γ-SNAP, and NSFmyc were incubated for 30 minutes at 4°C. The binding buffer contained 0.5 mmol/L ATPγS and 1 mmol/L EDTA. The NSFmyc was then immunoprecipitated with an excess of anti-myc, 9E10, antibody coupled to Protein G-Superose beads. Following immunoprecipitation, the beads were washed first in binding buffer and then with ATPγS/Mg (ATPγS/Mg Wash). The beads were specifically eluted with ATP/Mg (ATP/Mg Release). Material remaining on the beads was harvested by treatment with 200 mmol/L glycine/HCl, pH 2.7, O.5% Triton X-100 (Beads). All eluted proteins were analyzed by SDS-PAGE followed by Western blotting. Blots were probed with antisera directed against syntaxin 2, syntaxin 4, α-SNAP, and synaptobrevin 2. In Syb-2, BBE, detergent-solubilized bovine brain membrane extract was used in place of the HPE used in Syb-2, HPE.