FIG. 6.

BAF250a is required for GR-mediated transcriptional activation of chromatin MMTV. (A) Western blot analysis to monitor protein expression of transfected plasmids and to evaluate the extent of protein knockdown upon introduction of siRNA. Protein expression from SW-13/MMTV cells transfected with GR and BRG1 plasmid and specific siRNAs targeting BAF250a, BAF250b, BAF155, BAF170, or NTC (nontargeting control) was evaluated using the indicated antibodies. wt, wild type. (B) Four siRNA duplexes, specific for BAF250a, were used to evaluate the requirement of BAF250a in GR-mediated BRG1-dependent transcriptional activation of chromatin MMTV. Relative MMTV-luciferase expression levels in treated SW-13/MMTV cells cotransfected with expression plasmids for GR and BRG1 and the indicated siRNAs are shown. Luciferase activity was normalized to the total protein measured (measured in relative light units [RLU] per microgram of protein). NTC siRNA was used as a negative control. Values are shown as means plus standard deviations (error bars) from three experiments. Et-OH, ethanol. (C) BAF250a is not required for recruitment of BRG1-containing SWI/SNF to stably integrated MMTV. BRG1 remodeling complex is recruited to stably integrated MMTV in a GR-dependent manner in SW-13 cells independent of BAF250a protein, as demonstrated by ChIP analysis using anti-V5 antibody (α-V5). Purified immunoprecipitated DNA fragments were analyzed by real-time PCR using primer sets covering the MMTV promoter (Nuc-B) or 3′-coding region of the luciferase reporter (Luc-3′CR). Data are displayed as percentages of ChIP input. Normal IgG was used as a negative control. Data represented are from three independent experiments.