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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 6. ENaC subunits at the cell surface contain monoubiquitin but not polyubiquitin chains.

Figure 6

Panel A. Nedd4-2 or a control empty vector was transfected with ENaC subunits and HA-tagged ubiquitin as indicated into HEK293. Biotinylated surface proteins (panel above) were affinity purified and then immunoprecipitated with an anti-FLAG antibody and blotted with an anti-HA antibody (upper panel). Total cellular lysates were also immunoblotted with an anti-FLAG antibody (middle panel) and anti-Xpress antibody (bottom panel), the epitope tag attached to Nedd4-2. Panel B. and C. HEK 293 cells were co-transfected with increasing amounts of ENaC subunits (0, 5, 10, 15 μg of each subunit). The cells were treated with 1μM MG132 for 4 hours and biotinylated surface proteins were affinity purified and then immunoprecipitated with an anti-FLAG antibody and blotted with an anti-FK1 antibody (B) or anti-P4D1 (C). Total cellular lysates were also immunoblotted with these antibodies. The anti-polyubiquitin antibody FK1 detects ubiquitin chains only in cellular lysate and not in surface fractions whereas anti-P4D1 which identifies both monoubiquitin and polyubiquitin chains identifies ubiquitin in surface fractions.