Depletion of SMRT inhibits SRC-3 and ERα interaction, recruitment of SRC-3 to cyclin D1 enhancer and ERα transcriptional activity. A, MCF-7 cells were transfected with 20 nm control or SMRT siRNA and 48 h thereafter were treated with either vehicle (V) or 10 nm E2 for 60 min, followed by cell lysis and IP with ERα antibody. Levels of SRC-3 immunoprecipitated with ERα were assessed by Western blot (left). Five percent of the total cell lysates was reserved (INPUT), and Western blots were employed to assess SMRT, SRC-3, ERα, and actin expression (right). The experiment was performed three times, and a representative blot is shown. B and C, MCF-7 cells were transfected with 20 nm control or SMRT siRNA, and 48 h later, cells were treated with either vehicle (Veh) or 10 nm E2 for 45 min and subjected to ChIP assay using antibodies for ERα, SMRT, SRC-3, or IgG. Immunoprecipitated chromatin was quantitated by qPCR using primers for the cyclin D1 enhancer 2. Data represent an average ± sem of two to three independent experiments. D, HeLa cells were transfected with control siRNA or Sαβ2 siRNA before transfection with an ERE-E1b-Luc reporter gene and expression vectors for ERα, SRC-3, or vector control (−; pCR3.1) and treated with vehicle (Veh) or 1 nm E2. Values represent the average ± sem of three independent experiments. *, P < 0.01 in comparison with the respective E2-treated control values.