Figure 5
Phosphorylation of endogenous ERα in ERα (+) cell lines at S282, S294, and S559. (A) 107 MCF-7 and MCF-7-(LCC2) breast cancer and Ishikawa endometrial adenocarcinoma cell lines were cultured in medium supplemented with 10% FBS. Cells were lysed and total ERα was immunoprecipitated with α-p-S282, α-p-S294, or α-p-S559 antibodies for 3 hours. Immunoprecipitates were analyzed by Western blot for total ERα (B) S282 and S559 are phosphorylated following incubation of MCF-7 breast cancer cells with estradiol. MCF-7 breast cancer cells were cultured for 48 hours in phenol red free medium supplemented with 10% charcoal-stripped FBS. Cells were serum starved overnight prior to incubation with vehicle (veh) or 10-8M estradiol (E2). ERα was immunoprecipitated from lysates and Western blot analysis performed with α-pS282, α-pS294, or α-pS559, and with α-ERα. Substantial ligand-induced phosphorylation was detected at S282 and S559, with only modest ligand induced phosphorylation at S294.