Figure 4

ChIP-reChIP analyses identify coregulators that associate with AR/ERα on the RORA promoter. (A) SH-SY5Y cells were treated with 1 nM DHT and whole-cell lysates were prepared and immunoprecipitated with anti-AR or IgG antibody. The immunoprecipitated chromatin-antibody complex was then dissociated and washed to remove the antibody. The immunoprecipitated chromatin was then re-immunoprecipitated using anti-NCOA1, anti-NCOA5, anti-SUMO1, anti-FHL2, nonspecific IgG antibody, or no-second-antibody negative control. The enrichment of AR binding sites in the RORA promoter region in each re-ChIP sample was then determined using qPCR analysis (n = 3) and normalized with reference to the no-second-antibody control. (B) Gel electrophoresis analysis of PCR products using input chromatin, chromatin immunoprecipitated with anti-AR followed by anti-SUMO1 antibody, or IgG followed by IgG, as templates. PCR primers were designed to specifically amplify ARbs-I. ( C ) ChIP-reChIP analysis of coregulators associated with ERα binding sites in RORA promoter was conducted in the same manner as for AR binding sites using SH-SY5Y cells treated with 1nM E2. Error bar indicates SEM. **P <0.01, *P <0.05. AR, androgen receptor; ChIP-reChIP, sequential chromatin immunoprecipitation; DHT, 4,5α-dihydrotestosterone; E2, 17β-estradiol; ERα, estrogen receptor alpha; SEM, standard error of the mean.