Analysis of Pyk2 and PI3-K activation in estrogen-treated platelets. (A) Pyk2 and (B) p85/PI3-K were immunoprecipitated from gel-filtered platelets treated with 100 nM 17β-E2 (E2) for 60 or 180 seconds. Pyk2 was also immunoprecipitated from platelets preincubated with 10 μM PP1 for 15 minutes before treatment with 100 nM 17β-E2 for 60 seconds. Immunoprecipitated proteins were analyzed by immunoblotting with antiphosphotyrosine antibodies and then were reprobed with the same antibody used for immunoprecipitation (anti-Pyk2 or anti-p85/PI3-K, as indicated). Analysis of Akt phosphorylation was performed in resting platelets or in platelets treated with 100 nM 17β-E2 for 60 seconds or stimulated with 1 U/mL thrombin (Thr) or 2 μM U46619 for 180 seconds. Identical amounts of whole platelet proteins were analyzed by immunoblotting with anti–phospho-Akt-Ser473 or anti–phospho-Akt-Thr308 antibodies. (C) In parallel, corresponding aliquots of the samples were analyzed by immunoblotting with anti-Akt antibody. Results are representative of those of at least 3 separate experiments.