Fig. 3.

ERα signaling to FAK requires Gα_i/Gβ. A, Breast cancer cells were exposed for 20 min to 10 nm E2, in the presence or absence of the G protein inhibitor PTX (100 ng/ml), of the c-Src inhibitor, PP2 (0.2 μm), or of the PI3K inhibitor WM (30 nm), and Tyr³⁹⁷ FAK phosphorylation was assayed with Western analysis. B, Breast cancer cells were treated with E2 (10 nm) after transfection with dominant-negative Gα₁₃ or Gα_i constructs or siRNAs vs. Gβ₁. Gα₁₃, Gα_i, Gβ₁, actin, FAK, or phospho-FAK³⁹⁷ was assayed in cell extracts. C–E, T47-D cell protein extracts were immunoprecipitated with antibodies toward ERα, Gα_i1, or Gβ₁, and coimmunoprecipitation of Gα_i1 (C), ERα and ERβ (D), and ERα and Gα_i1 (E) was tested by Western analysis. F, Breast cancer cells were exposed for 20 min to 10 nm E2, in the presence or absence of the MAPK inhibitor PD98059 (PD; 5 mm), and Tyr³⁹⁷ FAK phosphorylation was assayed with Western analysis. CON, Control; IP, Immunoprecipitation. All experiments were performed three times, and representative blots are presented.