WASp is phosphorylated on tyrosine in platelets; effect of inhibitors. (A) Tyrosine phosphorylation of WASp in stimulated platelets. Western blots of WASp immunoprecipitates (10% SDS-PAGE) were probed with antiphosphotyrosine (4G10; upper panel) and, after stripping of the blot, anti-WASp antibodies (lower panel). WASp was tyrosine phosphorylated under basal conditions (lane 1) and underwent an increase in phosphorylation after stimulation by collagen (10 μg/mL) for 90 seconds (lane 2); CRP (3 μg/mL) for 90 seconds (lane 3); and thrombin (1 U/mL) for 30 seconds (lane 4). (B) The effect of inhibition of PLC activity on tyrosine phosphorylation of WASp. Western blots of WASp immunoprecipitates were probed with antiphosphotyrosine (4G10; upper panel) and, after stripping of the blot, anti-WASp (middle panel) and anticortactin (lower panel) antibodies. Basal conditions are shown in lane 1 and platelets stimulated with CRP (3 μg/mL, 90 seconds) are shown in lane 2. No significant effect on tyrosine phosphorylation induced by CRP occurred when platelets were preincubated with 5 μmol/L Ro31-8220 for 5 minutes and 40 μmol/L BAPTA-AM for 5 minutes (lane 3). (C) The effect of PI 3-kinase inhibitors on tyrosine phosphorylation of WASp. (D) The effect of tyrosine kinase inhibitors on phosphorylation of WASp. In (C) and (D), conditions are as in (A), with the exception that the probe for cortactin is not shown. One experiment is shown that is representative of 3.