Figure 7.
Aldosterone-induced NF-κB activation requires induction of SGK1. (A through D) mCCDcl1 were transfected with siRNA targeting SGK1 or scrambled RNA (A and B) or with SGK1 or eGFP (C and D). Cells seeded on plastic support (A and C) or on polycarbonate filters (B and D) were incubated in the absence (Ctl) or presence of 10−6 M aldosterone (Aldo) for 6 h, before protein (A and C) or RNA (B and D) extraction. (A) Representative immunoblot showing the efficient knockdown of SGK1 by siRNA. Bars represent densitometric analysis of immunoblots from aldosterone-treated cells transfected with siRNA, expressed as fold of stimulated scrambled RNA transfected cell OD (n = 2). (B) Silencing of SGK1 prevented the effect of aldosterone on NF-κB–dependent genes. IκBα, PAI-1, and IL-6 mRNA were detected by RT-PCR. Results are expressed as fold of control values set to 1. Bars are means ± SEM from six independent experiments. *P < 0.05 versus aldosterone-stimulated scrambled RNA. (C) Representative immunoblot showing that SGK1 protein abundance increased three-fold in cells transfected with SGK1 (n = 2). Bars represent densitometric analysis of immunoblots, expressed as fold of eGFP OD. (D) SGK1 overexpression increased IκBα mRNA abundance, detected by RT-PCR. Results are expressed as fold of control values set to 1. Bars are means ± SEM from four independent experiments. *P < 0.05 versus scrambled RNA. (E) mCCDcl1 cells grown on plastic support were treated or not (Ctl) with 10−6 M aldosterone (Aldo) for 6 h. Total protein extracts were immunoprecipitated with an anti-SGK1 antibody (see the Concise Methods section). Total cell lysates (Inputs) and immunoprecipitated proteins (IP: SGK1) were separated by 10% SDS-PAGE, and IKKβ and SGK1 were detected by immunoblotting. A representative blot from four independent experiments is shown.