Figure 3.

Effect of aldosterone and dDAVP on expression of luciferase γ-ENaC UTR constructs in mCCD cells. (A) mCCD cells were transfected with the original pGL3p-promoter (pGL3p) vector or chimeric variants, where original luciferase mRNA 5′- and/or 3′-UTRs were substituted by rat γ-ENaC 5′- and/or 3′-UTR. Eighteen-hour post-transfection cells were incubated for further 24 h under control conditions (without hormone; 0.1% ethanol) or with aldosterone (300 nM) or dDAVP (10 nM). UTR-dependent luciferase activity was measured 24 h after stimulation. Transfection efficiency was normalized to expression of co-transfected ‘Renilla’ luciferase and relative values were related to pGL3p and control (0.1% ethanol). Data represent mean ± SD (n = 6). *P ≤ 0.05 compared with control. (B) cDNA sequence of the entire 3′-UTR of rat γ-ENaC mRNA (Scnn1g, genbank NM017046) starting with the stop-codon (bold italics) is shown (nt 2049–2987). The AU-rich region deleted in luciferase chimeric constructs used for ARE-BP co-expression experiments is underlined and printed in bold. (C) Alignment of cDNA sequence of γ-ENaC mRNA 3′-UTR ARE of rat (NM017046) and mouse (NM011326).