Fig. 8.

Aldosterone promotes Sp1 recruitment to the R3 (−57/+438) subregion of the αENaC promoter without altering Sp1 protein levels. A: mIMCD3 cells were cultured in DMEM/F12 plus 10% charcoal-stripped serum and then treated with vehicle, 1 μM aldosterone, 1 μM spironolactone, or 1 μM aldosterone and 1 μM spironolactone for 2 h as indicated. The cells were harvested and chromatin was prepared. ChIP/qPCR assays (n = 3) with the anti-Sp1 antibody and primers to amplify the R3 subregion. The value for the final amplicon/input DNA obtained from the vehicle-treated cells was set as 1, and the aldosterone treatment values were normalized to it. Error bars indicate ± SE. *P < 0.05 vs. corresponding vehicle control. B: immunoblots of nuclear and cytoplasmic extracts from the vehicle- and aldosterone-treated cells were probed with antibodies directed against Sp1 or P84 (as a nuclear protein marker). Densitometric analysis was performed and the Sp1/P84 ratio was calculated. The value for vehicle-treated cells was set as 1, and the aldosterone treatment values were normalized to it (n = 3).