FIGURE 8.

NF-YC is sequentially recruited to the native αENaC promoter in an aldosterone-dependent manner followed by MR and SRC-1. For ChIP assays, when in the presence or absence (vehicle) of 10⁻⁷ m aldosterone, sheared chromatin from 293-MR cells was immunoprecipitated with anti-MR, anti-NF-YC, anti-SRC-1, anti-NF-YA, anti-NF-YB, or normal IgG. The coprecipitated DNA was amplified by quantitative real-time PCR, using primers to amplify the ENaC promoter containing MRE. At 45 min after aldosterone treatment, MR and SRC-1, interact with −1567/−1297 DNA segment containing MRE (bars 2 and 8). After additional 45 min, NF-YC sequentially interacts with −1567/−1297 DNA segment containing MRE (bar 12). Neither NF-YA nor NF-YB interacted with this region (bars 13–18). Immunoprecipitation with normal IgG (bars 4–6) was used as negative controls. Values are means ± S.D. for triplicates. Statistically significant differences as compared with control conditions are as follows: *, p < 0.05 versus 0 min; **, p < 0.01 versus 0 min; ***, p < 0.001 versus 0 min; ^#, p < 0.05 versus IgG; ^##, p < 0.01 versus, IgG; and ^###, p < 0.001 versus IgG.