FIG. 2.

Aldosterone induces the activation of Nrf2 in LLC-PK1 cells. (A) Dose dependency of Nrf2-activation. Nrf2-DNA binding was measured by EMSA in nuclear fractions, incubated in the absence (0) and presence of the indicated aldosterone concentrations for 30 min. (B) Kinetics of aldosterone (100 nM) induced Nrf2-DNA binding in nuclear fractions as measured by EMSA. To determine the specificity of the Nrf2-DNA complex, the control nuclear fraction (0 h incubation) was incubated in the presence of 100-fold molar excess of unlabeled oligonucleotide containing the consensus sequence for either Nrf2 (cold) or SP-1 (SP-1) before conducting the binding assay. The nuclear presence of Nrf2 (95–110 kDa) was evaluated by Western blot in LLC-PK1 cells that were incubated with (C) 10 nM aldosterone and (D) 100 nM aldosterone. α-tubulin was measured as a loading control. Results are shown as ratio of Nrf2/α-tubulin. (A, B) Shown are mean values±SEM, *p≤0.05 versus time point 0, tested by ANOVA with subsequent post-hoc comparisons by Scheffé. (C, D) Shown is the median±the standard error of the median, *p≤0.05 versus time point 0, evaluated by Mann–Whitney test. EMSA, electrophoretic mobility shift assay.