Collagen and CRP stimulate phosphorylation of PLCγ1 on Tyr 783 in murine platelets. (A) Washed platelets were stimulated with 10 μg/mL CRP or collagen for 30 seconds and phosphorylation was measured as described in “Materials and methods.” In brief, proteins were precipitated with anti-PLCγ1 or PLCγ2 antibody, resolved by 8% SDS-PAGE, transferred to PVDF membranes, and immunoblotted with antibodies against phosphotyrosine 783 in PLCγ1, phosphotyrosine, PLCγ1 or PLCγ2. (B) Before stimulation, washed platelets were incubated with DMSO, 20 μM PP2, or 100 nM wortmannin. Immunoprecipitation with anti-PLCγ1 antibody and immunoblotted with antiphosphotyrosine 783 in PLCγ1 or anti-PLCγ1 antibody were performed as described for panel A. The data are representative of at least 2 experiments.