FIGURE 2.

Proteolytic cleavage of wild-type and mutant channels mutated on cytoplasmic lysines. A, Hek293 cells were transiently transfected with either wild-type (W) or cytoplasmic lysine mutant (K) ENaC. 24 h after transfection, cells were biotinylated, recovered with streptavidin-Sepharose and analyzed by SDS-PAGE/Western blotting using anti α-, or γ-ENaC antibodies as indicated. fl: full-length α- or γ-ENaC; arrow: cleaved α- or γ-ENaC. α- and γENaC antibodies crossreact with endogenous proteins, as shown in lane 1. In our previous work we have provided evidence that these do not represent endogenous ENaC. B, quantification of the ratio of cleaved to full-length αENaC (as described under “Experimental Procedures”), normalized to wild-type ENaC (condition 2), and displayed as mean ± S.E. (n = 3 experiments; *, p < 0.05 versus wt ENaC (2) as determined by Student's t test).