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. Author manuscript; available in PMC: 2009 May 1.
Published in final edited form as: Am J Physiol Renal Physiol. 2008 Mar 5;294(5):F1157–F1165. doi: 10.1152/ajprenal.00339.2007

Figure 3. Inactivation of the catalytic domain of Nedd4-2 abolishes its effect on ENaC.

Figure 3

Panel A. Active or inactive Nedd4-2 (Nedd4-2CS) or a control empty vector was transfected with ENaC subunits as indicated into HEK293 cells and biotinylated surface proteins and total cellular lysates were blotted with an anti-FLAG antibody, or an actin antibody. The data demonstrates that Nedd4-2 CS does not affect surface expression of ENaC. Actin was immunoblotted in total and surface fractions to confirm that intracellular proteins are excluded in affinity purified surface protein preparations. Panel B. Active or inactive Nedd4-2 (Nedd4-2CS) or a control empty vector were transfected with ENaC subunits and HA-tagged ubiquitin (HA-Ub) as indicated into HEK293 cells. ENaC subunits were immunoprecipitated with an anti-FLAG antibody and immunoblotted with anti-HA to detect ubiquitinated ENaC. Ubiquitinated proteins appear as high molecular mass smear. Compared to active Nedd4-2, Nedd4-2CS has no effect on ubiquitination of ENaC in HEK293 cells. Panel C: Adenoviral vectors expressing active or inactive Nedd4-2 (Nedd4-2CS) or a control empty virus were transduced into M-1 cells. Compared to active Nedd4-2, Nedd4-2CS has no effect on amiloride-sensitive Isc in M-1 cells. (*P < 0.05; Dunn’s pairwise multiple comparison, SigmaStat; n = 6; mean ± SD). Panel D. Representative western blot of transduced M-1 cells demonstrate relatively equal expression of the Nedd4-2 isoforms.