The role of platelet signaling cascades in IKK activation and SNAP-23 phosphorylation. (A) Mouse platelets were preincubated with BAPTA/AM (40 μM) or EGTA (1 mM) for 3 minutes and then stimulated with either thrombin (0.1 U/mL) or thapsigargin (1 μM) for 3 minutes, and subjected to immunoblotting with anti-phospho-Ser95 (pSer-95) and anti-SNAP-23 antibodies. (B) Mouse platelets were preincubated with different inhibitors: 10 μM, rottlerin; 1 μM, Gö6976; and 1 μM, RO-31-8220 and PLC inhibitor (U-73122, 5 μM) or its inactive isomer (U-73343, 5 μm) for 5 minutes and then stimulated with thrombin (0.1 U/mL, 3 minutes). Extracts from these platelets were subjected to immunoblotting with anti-phospho-Ser95 (pSer-95) and anti-SNAP-23 antibodies. (C) Mouse platelets were preincubated with the indicated PKC inhibitors then activated with thrombin (0.1 U/mL, 3 minutes) and extracts from them were subjected to immunoblotting with anti-pSer32-IκB (pIκB) or anti-IκB antibodies. (D) Mouse platelets were labeled with [³²P] H₃PO₄ then preincubated with the indicated inhibitors (1 μM, RO-31-8220 or 5 μM BMS-345541) prior to stimulation with thrombin (0.1 U/mL). The reactions were stopped with SDS sample buffer, the total platelet proteins were separated by SDS-PAGE, and the phospho-proteins were visualized by phosphorimaging.