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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 8.

Figure 8

The effect of Nedd4-2 isoforms and proteasomal inhibitors on ENaC function: Panel A. ENaC subunits were expressed with and without HA-tagged Ubiquitin (HA-Ub) in HEK293 cells and cells were treated with or with-out 1μM MG132 or 100 μM chloroquine for 4 hours as indicated. Ubiquitinated ENaC subunits were detected by immunoprecipitation with anti-Flag followed by immunoblotting with anti-HA antibody. There appeared to be more ubiquitinated ENaC detectable in the presence of MG132. Panel B. M-1 cells grown in filters were treated with or without 1μM MG132 and Isc measured in Ussing chambers. Isc was expressed as fold change compared to the control value corresponding to the same time point. Currents increase significantly within an hr of MG132 and reaches a peak between 2-3 hr (**P < 0.001; *P < 0.05 Tukey’s pairwise multiple comparison, SigmaStat; n = 8 from three different experiment; means ± SE). Panel C. M-1 cells were transduced with adenovirus expressing Nedd4-2 or empty virus and Isc measured in Ussing chambers under control conditions and after treatment with MG132 or chloroquine for 2 hr. M-1 cells overexpressing Nedd4-2 had significantly lower currents as compared to empty virus. MG132 but not chloroquine partially reverses the effect of Nedd4-2 on Na+ transport (*P < 0.05 between vehicle and MG132 ; ¶P < 0.05 between vehicle and chloroquine; Tukey’s pairwise multiple comparison, SigmaStat; n = 12 from 3 experiments; mean ± SE).

Panel D. M-1 cells were transduced with adenovirus expressing each of the Nedd4-2 isoforms and Isc measured in Ussing chambers under control conditions and after treatment with MG132 or chloroquine for 2 hr. M-1 cells overexpressing Nedd4-2ΔC2 had lower currents compared to Nedd4-2 and Nedd4-2ΔWW2,3. MG132 partially reverses the effect of Nedd4-2 and Nedd4-2ΔWW2,3, but not Nedd4-2ΔC2, on Na+ transport (*P < 0.05 between vehicle and MG132 ; ¶P < 0.05 between vehicle and chloroquine; Tukey’s pairwise multiple comparison, SigmaStat; n = 16 from 3 experiments; mean ± SE).