PECAM-1 dimerization results in its tyrosine phosphorylation. Washed platelets (1 × 109/mL) were incubated at 37°C with the following agonists in the presence of 2 mmol/L CaCl2, 1 mmol/L MgCl2, and 100 μg/mL fibrinogen: (1) buffer (in the presence of stirring), (2) 7 μmol/L TRAP for 5 minutes under stirring conditions (fully aggregated), (3) 10 μg/mL normal mouse IgG1 F(ab′)2fragments for 30 minutes (stirred), and (4) 10 μg/mL PECAM-1.2 F(ab′)2 for 30 minutes (stirred). After detergent lysis, immunoprecipitations (IP) were performed using either normal mouse IgG1 (NM, left panel) or PECAM-1.3 (right panel). Bound proteins were resolved by 12.5% SDS-PAGE and analyzed by immunoblotting using an HRP-conjugated antiphosphotyrosine antibody (PY-20). Arrows indicate the positions of tyrosine phosphorylated PECAM-1 and the heavy chain of IgG.
