Figure 5.

PCNA associates with endogenous estrogen-responsive genes. (A) Sheared chromatin from MCF-7 cells, which had been treated with ethanol vehicle (white bars) or 10 nM E₂ for 2 h (gray bars), was immunoprecipitated with an ERα- or PCNA-specific antibody. DNA was isolated and real-time PCR was performed in triplicate to monitor the association of ERα and PCNA with the region of the pS2 gene containing an imperfect ERE or a region 2.8-kb upstream of the pS2 ERE (Control). Standard curves were derived for each primer set and the relative copy number for each sample was obtained based on the standard curve. Data from four independent experiments are expressed as the mean ± SEM. A significant change in the copies associated induction in the presence of E₂ was determined by Student's t-test and is indicated by an asterisk (*, P < 0.05). (B) Sheared chromatin from MCF-7 cells, which had been treated with ethanol vehicle or 10 nM E₂ for 15, 45 or 120 min, was immunoprecipitated with an ERα- or PCNA-specific antibody or non-specific IgG. DNA was isolated and subjected to PCR amplification to monitor the association of ERα and PCNA with the ERE-containing region of the oxytocin gene or the non-estrogen-responsive 36B4 gene. Ten percent of input DNA was included as a control. PCR products were run on 1.5% agarose gels, stained and visualized using UV light. Results are representative of three independent experiments.