Physical association of prohibitin and E2F1 is required for the repressive function of estrogen antagonists. MCF7 cells were stably transfected with pCR3.1 E2F1 (AA304–357) or pCR3.1 E2F1 (AA263–303), both tagged with Gal4. Expression of the transfected E2F peptides was confirmed by immunoblot analysis (C). (A) Whole-cell extracts, or proteins from cell extracts immunoprecipitated using anti-Gal4 (for the E2F mutants) or control (anti-Myc or -E2F) antibodies, were separated electrophoretically, then identified by immunoblotting using an anti-prohibitin antibody, or anti-tubulin antibody as a loading control (upper panel). The experiments were repeated in a reciprocal fashion (lower panel). (B) The E2F peptide-expressing MCF7 cells were transfected with an E2Luc reporter and treated with ethanol (vehicle control) or estrogen antagonists. Cells were harvested for luciferase activity assay and β-galactosidase assay (transfection control). Luciferase activity calculations are normalized for β-galactosidase activity.