PLC activation is normal in Vav1-deficient mouse platelets.

(A) For analysis of phosphatidic acid (PA) levels, platelets from control and Vav1-deficient mice were labeled with [³²P]orthophosphoric acid, and stimulated with the indicated concentrations of CRP or thrombin for 5 minutes at 37°C and PA extracted as described in “Materials and methods.” PA levels were detected by autoradiography of the TLC plate. (B) For analysis of pleckstrin phosphorylation, platelets from both control and Vav1^−/− mice were labeled with [³²P]orthophosphoric acid, and stimulated with the indicated concentrations of CRP or thrombin for 2 minutes at 37°C. Samples were separated by SDS-PAGE and the gel was dried and autoradiographed. Pleckstrin appears as a strongly phosphorylated band at approximately 45 kDa. (C) PLCγ2 was immunoprecipitated from control and Vav1^−/− mouse platelets stimulated with CRP at the indicated concentrations for 60 seconds. The resulting immunoblots were probed for phosphotyrosine (anti–p-Tyr), stripped, and reprobed for PLCγ2. This procedure was repeated for SLP-76 (lower panels). One experiment representative of 3 is depicted.