![Figure 5. 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.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/121/22/10.1182_blood-2012-11-470468/5/4567f5.jpeg?Expires=1722709847&Signature=q2JoeYv20~0CqbdvG2opG0F8Y4KOnxLKSdmDVqVVlyir3j0UtKe21wbBu78k~077rOSoybx~k83B5d0wr-bfgFjTY1Iuy9Kr2QzNzMZPDmFYRt2nhjOpSedWMu5CmUr~POjYTRU8LAtbSzw3fYhH4IjdOpEpM-YHhMXiqP63sU446VM~0SxPb6fAsk4wr4ZIpgZLU34UoD3-aIcYSS6LHgz~f65RuGK4v5j5SZtZLiU-pU4aBrtpPNSqLlf~TKTkXCoUOVlppNg0Jk8QjdBA-dWnsqDqLPKKiivvuFvQcMiGdG9y8iSEyrADRB~eQ8U3yIXqDwxmnYMBwpIT2cWPMA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
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.
