Phosphorylation of ephrinB1 during adhesion and aggregation. (A) Platelets were preincubated with 10 μM PP2, 10 μM PP3, or vehicle for 10 minutes at room temperature and then activated with GST-EphB1 (3 μg/mL) for 30 minutes. EphrinB1 was immunoprecipitated and then immunoblotted with the antiphosphotyrosine antibodies PY20 and 4G10 or with anti-ephrinB1. The results shown are representative of 2 experiments. (B) Platelets were preincubated for 15 minutes with either PP2 or PP3 and then stimulated for 20 minutes with GST-EphB1 or GST-ephrinA4 in fibrinogen-coated wells. The results shown are the mean ± SEM for 4 experiments. (C) Platelets were incubated with an agonist for 5 minutes under conditions allowing aggregation to occur or with GST-EphB1 for 30 minutes as a control, after which ephrinB1 was immunoprecipitated and then immunoblotted with either PY20 and 4G10 or with anti-ephrinB1. Final concentrations: PMA 100 nM, ADP 20 μM, SFLLRN 50 μM, convulxin 1 μM, U46619 5 μM, ionomycin 5 μM, and GST-EphB1 5 μg/mL. The results shown are representative of 3 experiments. The same results were obtained when the incubation with the agonists was extended to 30 minutes.