Formation of an ERβ-associated signaling complex in estrogen-treated platelets. (A) Platelets were treated with buffer or 100 nM 17β-E2 for the indicated times, and ERβ was immunoprecipitated with specific antibodies. Immunoprecipitated proteins were analyzed by immunoblotting with anti-Src antibody and were reprobed with anti-ERβ antibody. (B) Immunoprecipitates obtained with anti-ERβ antibodies from resting platelets or platelets incubated with 100 nM 17β-E2 for 60 and 180 seconds, together with aliquots of whole platelet lysates (WCL), were analyzed by immunoblotting with antibodies against pSrc-Tyr416, Pyk2, p85/PI3-K, FAK, Syk, and ERβ, as indicated on the left. (C) ERβ was immunoprecipitated from resting or 17β-E2–stimulated platelets (E2; 100 nM, 60 seconds) preincubated for 15 minutes in the absence or presence of 10 μM PP1 or 50 nM wortmannin. Immunoprecipitated proteins were analyzed by immunoblotting with antibodies against Pyk2, p85/PI3-K, Src, and ERβ. Results are representative of those of at least 3 different experiments.