17β-E2–dependent Src kinase activation. (A) Gel-filtered platelets were incubated with buffer or with increasing concentrations of 17β-E2 (E2: 1, 10, 100, and 1000 nM) for 30 seconds. (B) Gel-filtered platelets were incubated with buffer or with 100 nM 17β-E2 (E2) for increasing times (0, 30, 60, and 180 seconds.) Platelets were lysed, and identical aliquots from each sample were simultaneously analyzed by immunoblotting with specific antibodies against pSrc-Tyr416 (Src PY 416) or against Src. Platelet stimulation with 100 nM 17β-E2 for 30 seconds was performed in the presence of 10 μM ICI 182 780, an ER antagonist. Results are representative of those of several different experiments.