FIG. 3.

BAF57 is critical for AR activity. (A) MCF7 (lane 1), BT549 (lane 2), LNCaP (lane 3), and CV1 (lane 4) cells were harvested, lysed, and subjected to SDS-PAGE. Immunoblots were performed with antibodies to BAF57 and CDK4 (loading control). (B) MCF7, CV1, and BT549 cells (2 × 10⁵) were transfected with 0.5 μg of pARR2-Luc reporter, 0.5 μg of pSG5AR, and 0.25 μg of pTK-Renilla luciferase (to normalize for transfection efficiency). Posttransfection, cells were stimulated with either 0.1% EtOH vehicle or 0.1 nM DHT for 24 h. Cells were harvested, lysed, and monitored for luciferase activity by utilizing the Dual Luciferase Assay Reporter System. AR activity is presented as the fold activation by ligand (DHT) versus EtOH. Averages and standard deviations are shown. (C) BT549 cells either untransfected (lane 1) or transfected as described above with pGreenLantern (indicating transfection) and pSG5AR (lane 2) were harvested 24 h posttransfection. Immunoblot analysis demonstrates AR expression in transfected BT549 cells. (D) To elucidate BAF57 function on AR reporter activity, BT549 cells (2 × 10⁵) were transfected with 0.5 μg of pARR2-Luc reporter, 0.5 μg of pSG5AR, 0.25 μg of pTK-Renilla luciferase, and either 2.25 μg of parental vector or pBabe-WTBAF57. Posttransfection, cells were treated with either 0.1% EtOH vehicle or 0.1 nM DHT for 24 h. Cells were harvested and lysed, and the luciferase activity was measured by using the dual Luciferase Assay Reporter System. Ligand-induced AR activity in the presence of vector was set to 100, and the relative luciferase activity is shown. (E) BT549 cells (2 × 10⁵) were transfected with 0.5 μg of pGreenLantern GFP (transfection control), 0.5 μg of SG5AR, and either 2.25 μg of vector (lane 1) or BAF57 expression plasmid (lane 2). Immunoblotting was performed for AR and GFP at 24 h posttransfection.