Tyrosine phosphorylation of Vav1.

(A-B) Human platelets were stimulated with CRP (1 μg/mL) as shown in (A) or with thrombin (thr; 1 U/mL) as shown in (B), and lysis buffer added at the indicated time point. Vav1 was immunoprecipitated from these samples and analyzed by immunoblotting with monoclonal antiphosphotyrosine antibody 4G10 (anti–p-Tyr). Immunoblots were then stripped and reprobed with an antibody to Vav1 (lower panel). (C) Human platelets were incubated with the PI 3-kinase inhibitors, wortmannin, or Ly294002, or the Src kinase inhibitor PP1, as described in “Materials and methods.” (D) CRP (1 μg/mL)–induced tyrosine phosphorylation of Vav1 in platelets from mice deficient in the tyrosine kinase Syk were analyzed at the indicated time points as described above.