Fig. 5. Rb is required for the prohibitin/Brg-1/Brm-mediated transcriptional repression. (A) Saos2 cells (lacking Rb) and C33A cells (Brg-1 and Brm deficient, and expressing a mutant Rb that cannot bind to Brg-1) were transfected with pSVECGCAT. The basal CAT activity was not affected by the co-transfection of GAL4–prohibitin, Brg-1 and Brm in Saos2 cells (lanes 1–4). The activity of pSVECGCAT was repressed when prohibitin was co-transfected with Brg-1 or Brm in C33A cells, while prohibitin, Brg-1 or Brm alone did not affect the activity (lanes 5–10). (B) Saos2 cell lysate was immunoprecipitated using either Brg-1, Brm or prohibitin antibodies followed by immunoblotting using prohibitin or Brg-1 and Brm antibodies. A c-Myc antibody was used as a negative control for the immunoprecipitation and a p38 antibody for the immunoblot analysis. (C) CHIP assay using the same Saos2 cell extracts shown in (A), which were transfected with pSVECGCAT and GAL4–prohibitin as indicated. The DNA recovered from immunoprecipitation by Brg-1, Brm, prohibitin or p38 (as negative control) antibodies was amplified by PCR, using primers against a region of the CAT gene. Co-transfection of the GAL4–prohibitin expression vector resulted in a PCR product using DNA recovered from the Brg-1, Brm or prohibitin immunoprecipitates, indicating that GAL4–prohibitin, Brg-1 and Brm are recruited to this promoter, which carries a GAL4-binding site. Direct PCR using the cell extracts without immunoprecipitation detected comparable amounts of the CAT gene from both extracts tested, demonstrating the equal transfection efficiency. PCR failed to detect the CAT gene in the p38 immunoprecipitates, indicating the specificity of the CHIP assay. (D) Saos2 cells were transfected with pSVECGCAT. Co-transfection of GAL4–prohibitin or Rb alone failed to repress the transcriptional activity of the reporter (lanes 1, 2 and 3). CAT activity was repressed by co-transfection of GAL4–prohibitin plus Rb expression vectors (lane 4), and this repression was reversed by co-transfection of dominant-negative Brg-1 (BJ5Brg1 K-R) or dominant-negative Brm (CGBrm NTP) (lanes 5–8).