(A through D, upper panels) Tyrosine phosphorylation of WASP in platelets stimulated by collagen (50 μg/mL), thrombopoietin (TPO; 100 ng/mL), or thrombin (1 U/mL) at the time intervals indicated. Platelets were lysed by the addition of an equal amount of a buffer containing 2% Triton X-100 before and after exposure to collagen, TPO, or thrombin. To prevent platelet aggregation, platelets were treated with RGDS (200 μmol/L), except in lanes 1 through 3 in (D). For experiments shown in (D), we used lysis buffer containing 0.2% SDS and sonicated the lysates. WASP was immunoprecipitated with the polyclonal anti-WASP antiserum (503). Immune complexes were resuspended in SDS sample buffer and divided into two. Proteins were separated by 10% SDS-PAGE and transferred onto PVDF membranes. One immunoblot was probed with antiphosphotyrosine antibodies and bands were visualized by chemiluminescence (upper panels, A through D). The other blot was probed for WASP (lower panels, A through D).