Estradiol-induced FKHR activation and Erα–FKHR complex assembly in MCF-7 cells. Model of ERα nuclear export. Quiescent MCF-7 cells were used. (a) Cells were left unstimulated or stimulated for 5 min with 10 nM estradiol in the absence or presence of LY294002 (at 5 μM; Qbiogene). Lysates were analyzed for FKHR phosphorylation using the anti–P-Ser-256–FKHR antibody (bottom). The nitrocellulose filter was then reprobed with anti-FKHR antibody (top). (b) The cells were transfected with the indicated plasmids (FKHR wt or FKHR AAA) then left unstimulated or stimulated for 30 min with 10 nM estradiol. Lysates were immunoprecipitated with anti-ERα antibody. Immunocomplexes were immunoblotted using the antibodies against the indicated proteins. (c) The model of ERα nuclear export is depicted. Estradiol stimulates the PI3K–AKT pathway, leading to FKHR phosphorylation and triggering the functionally associated export of FKHR–ERα. This export results in stimulation of DNA synthesis.