Signaling components involved in PKD activation.
Washed human platelets (1 × 109/mL) were pretreated with 1 μM Ro 31-8220 (Ro), 10 μM PP1, 100 nM wortmannin (Wm), and 20 μM BAPTA-am (BAPTA), prior to activation with 30 nM PMA (panel A) or 12 nM convulxin (Cvx; panel B), for 1 minute at 37°C. Samples from whole cell lysates were analyzed by Western blotting using pS916 following lysis of the cells with 2 × sample buffer. Results are representative of the mean ± SEM of 3 experiments. The remaining cells were lysed and immunoprecipitated using the pan sc937 antibody. PKD activity immunoprecipitated from whole cell lysates was measured by in vitro kinase assay using syntide-2 as substrate. Results shown are the mean ± SEM of 3 independent experiments with duplicate samples from each. (C) Platelets were lysed and analyzed by SDS-PAGE and Western blotting with the pS916 antibody and the Western blots were reprobed with a pan–C-terminal PKD antibody (pan sc639). One experiment representative of 3 independent experiments is shown. Platelets were labeled with 32P-orthophosphate for 1 hour prior to stimulation with 0.1 U/mL thrombin (Thr) or 0.1 U/mL thrombin preincubated with 100 nM wortmannin (Wm). At various time intervals samples were removed to determine the degree of pleckstrin phosphorylation, as described in “Materials and methods.” Results are shown as the mean ± SEM of 3 independent experiments.
