FIGURE 4.

Limited proteolysis using trypsin on ENaC channels co-expressed with Usp2-45 and/or DynK44A. A, Hek293 cells were transiently transfected with either wild-type or furin mutant (fu) ENaC together with Usp2-45 WT or with DynK44A, or with Nedd4-2 WT. 24 h after transfection, cells were treated with 1 μg/ml trypsin for 5 min at 37 °C, and biotinylation was performed to recover proteins present at the cell surface. Recovered proteins were run on a SDS/PAGE and analyzed by Western blot as indicated. B, in 4 individual experiments the cleaved 30-kDa α fragment and full-length αENaC was quantified. The ratio of cleaved to full-length α is displayed. *, p < 0.05 versus ENaC fu; ***, p < 0.001, ##, p < 0.01 versus wt ENaC (8) (n = 4).