Analysis of Pyk2 phosphorylation induced by integrin α2β1. Adhesion-induced Pyk2 phosphorylation was evaluated on whole-platelet lysates by immunoblotting with anti–phospho-Pyk2(Tyr402) Ab (top rows). Subsequent staining with anti-tubulin or anti-pleckstrin Ab is reported in the bottom rows as a control for equal loading. (A) Platelet adhesion to monomeric collagen (i) or GFOGER peptide (ii) was performed for the times indicated on the bottom. Samples of nonadherent platelets were collected after 60 minutes. (B) Analysis of Pyk2 phosphorylation in wild-type and PI3Kβ^KD murine platelets adherent to monomeric collagen for the 10, 30, or 60 minutes. (C) Effect of platelet incubation with BAPTA-AM (20μM, 30 minutes) or 2-APB (100μM, 10 minutes) on integrin α2β1–induced Pyk2 phosphorylation. Platelet adhesion was performed for 60 minutes. None indicates control platelets treated with DMSO; NA, nonadherent, untreated platelets. (D) Analysis of Pyk2 phosphorylation in platelets from wild-type (WT) and PLCγ2–knockout (PLCγ2 KO) mice after adhesion to monomeric collagen for 30 and 60 minutes.