Figure 3.

RNA Silencing of E2F1 increases p65/p50 heterdimer binding to ICAM-1 promoter. A. ChIP assay was performed with cell lysates from DU145/sh-Con and DU145/sh-E2F1 cells. The chromatin was immunoprecipitated with anti-E2F1 and anti-p65 antibodies or normal IgG which served as a negative control. A pair of primers flanking the κB-1 binding site within the ICAM-1 promoter was used in PCR. PCR for the E2F1 binding site within the CDC2 promoter served as a positive control for detecting E2F1 binding activity. B. Real-time PCR was employed to the ChIP assay in (A). Relative occupancy values were calculated by determining the apparent immunoprecipitation efficiency (ratios of the amount of immunoprecipitated DNA to that of the input sample) and normalized to the level of p65 binding with ICAM-1 promoter in DU145/sh-Con cells, which was defined as 1.0. C. The relationship among E2F1 and p65/p50 heterodimer in DU145/sh-Con and DU145/sh-E2F1 cells was examined by coimmunoprecipitation analysis. Anti-p65 antibody was used for immunoprecipitation. The amounts of E2F1, p65 and p50 in the immunoprecipitates were detected by Western blot with the indicated specific antibodies.