Loss of SNAP23 results in increased amounts of the syntaxin1A (Stx1A)–VAMP2–SNAP25 complex. (A) In vitro competition experiment demonstrating that SNAP23 and SNAP25 compete with each other for binding to Stx1A and VAMP2. His-SNAP23 (0, 0.6, 5, and 10 µM), His-Stx1A, and His-VAMP2 (0.6 µM each) were mixed to form recombinant SNARE complex and then combined with GST-SNAP25 (0.6 µM) and glutathione beads. The beads were analyzed by SDS-PAGE and immunoblotting. (B) Scheme of SNAP23 competing with SNAP25 for binding to Stx1A and VAMP2 in the GST pulldown assay. (C) SNAP23 did not coimmunoprecipitate with SNAP25. Islet lysates from wild-type mice were immunoprecipitated (IP) with antibodies against SNAP25 and control IgG. (D) Coimmunoprecipitation of Stx1A and VAMP2 in SNAP25 immunoprecipitates from control (Ctrl) and BcKO islet lysates. The asterisk indicates the IgG light chain. (E) VAMP2 intensity in the SNAP25 immunoprecipitates from BcKO relative to control islet lysates. The data are representative of three independent experiments. (F) KCl-induced secretion of hGH from MIN6 cells transfected with hGH together with control siRNA, siRNA against SNAP23, or siRNA against SNAP25, as measured by a batch release assay and an ELISA. The data are representative of five independent experiments. (G and H) Coimmunoprecipitation of Stx1A and VAMP2 in SNAP25 immunoprecipitates from control siRNA–treated or SNAP23 siRNA–treated MIN6 cell lysates. The asterisk indicates the IgG light chain. (H) VAMP2 intensity in the SNAP25 immunoprecipitates from SNAP23-knockdown MIN6 relative to control MIN6 cell lysates. The data are representative of three independent experiments. Data are mean ± SEM. Significance was calculated by the two-tailed paired Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.