IKK-mediated phosphorylation of SNAP-23 is crucial for SNARE complex formation. Mouse (A-B) and human (C-D) platelets were incubated with IKK inhibitors (BMS-345541, 5 μM) (A-C); (TPCA-1, 0.5 μM) (B-D) and stimulated with thrombin (0.1 U/mL, 3 minutes). Wild-type (WT) and IKK-β−/− mouse platelets were stimulated with thrombin (0.1 U/mL) (E). Platelet lysates were precleared and then incubated with anti-syntaxin-11. Immunoprecipitates were separated by SDS-PAGE and immunoblotted using antibodies to syntaxin-11, SNAP-23, phosho-Ser/Thr/Tyr (pSer/Thr/Tyr), phospho-Ser95 (pSer-95), and VAMP-8 as indicated. (F) SNARE-bearing vesicles, t- and v-SNARE were reconstituted as described in our “Methods” section. t-SNARE vesicles were incubated with 0.5 mM ATP, 10 mM MgCl2, and increasing amounts of IKKβ (0-1.0 μg/reaction) at RT. After 60 minutes, v- and t-SNARE vesicles were mixed and fusion was monitored at 37°C. After 60 minutes, n-dodecyl-β-d-octylglucoside was added to obtain the maximum NBD fluorescence and fusion was calculated as the percent of that maximum. (G) An aliquot of the t-SNARE mixture from some of the reactions was subjected to immunblotting using anti-phospho-Ser95 (pSer-95) and anti-SNAP-23 antibodies. The data are representative of 2 replicates.
