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. 2009 Jul 17;5(7):e1000567. doi: 10.1371/journal.pgen.1000567

Figure 1. BAF is essential for MMTV promoter activity in T47D-MTVL cells.

Figure 1

(A) Cells were transfected either with siRNA against Brg1, Brm , or both siRNAs combined as indicated. After 48 h the medium was replaced by fresh medium without serum. After one day in serum-free conditions, cells were lysed and the levels of Brm, Brg1, PR and tubulin were determined by Western blotting. C, control siRNA. (B) Cells were transfected with Control, Brg1 and Brm siRNAs as described in (A). After one day in serum-free conditions, cells were incubated with 10 nM R5020 for 8 hs and total RNA was prepared, cDNA was generated and used as template for real time PCR with luciferase oligonucleotides. The values represent the mean and standard deviation from 3 experiments performed in duplicate. (C) Cells were transfected with Control, Brg1 and Brm siRNAs as described in (A) and treated with 10 nM R5020. cDNA was generated and used as template for real-time PCR using specific c-Fos, c-Myc, Cyclin D1 and GAPDH primers. The values represent the mean±SD of 3 experiments performed in duplicate. (D) Cells were transfected with Control and BAF57 siRNAs as described in (A) and treated with 10 nM R5020. Left: BAF57 levels were analyzed by Western blotting. Right: RNA was extracted, cDNA was generated and used as template for real time PCR with luciferase oligonucleotides. The values represent the mean±SD of 2 experiments performed in duplicate. (E) Experiments similar to those shown in (D), but cells were transfected with Control and BAF250 siRNAs as described in (A) and (D). The values represent the mean±SD of 2 experiments performed in duplicate. (F) Cells were lysed and immunoprecipitated either with α-BAF57 antibody or with normal rabbit IgG as a negative control (IgG). The immunoprecipitates (IP) were analyzed by western blotting with non-discriminating Brg1/Brm antibody, α-BAF155 or α-BAF170 specific antibodies. (G) Cells were untreated (0) or treated for 5, 30 or 60 min with 10 nM R-5020 and subjected to ChIP assays with α-PR, α-Brg1, α-Brm, α-BAF57, α-BAF250, α-BAF180 and α-BAF200 specific antibodies. The precipitated DNA fragments were subjected to PCR analysis to test for the presence of sequences corresponding to the MMTV nucleosome B. Input material (1%) is shown for comparison. A representative of three independent experiments is shown.