Fig 4. Inhibition of ENaC by H-Ras is not mediated by Nedd4-2.

A, I_{ami (normalized)} in FRT cells transfected with wild-type α-, β-, and γ-ENaC (αβγ) subunits or with ENaC comprising one truncation mutant (α-ENaC mutant that is truncated at amino acid Pro⁶⁴⁶ (α_T); β-ENaC mutant that is truncated at amino acid Cys⁵⁹⁴ (β_T); or γ-ENaC mutant that is truncated at amino acid Phe⁶¹⁰ (γ_T)). The solid bar indicates that the monolayer were co-transfected with H-Ras^G12V. B, I_{ami (normalized)} in FRT cells co-transfected with wild-type ENaC subunits, and with pcDNA3.1 or pSM2c empty vector (control for shRNA-treated group) or with H-Ras^G12V together with or without an shRNA directed against Nedd4-2 (shNd4-2) in pSM2c as indicated. C, upper panel, Immunoblot analysis of membrane α-ENaC in HEK293 cells transfected with HA (N-terminus) and V5 (C-terminus) tagged α-ENaC (_HAα_V5) and β- and γ-ENaC with FLAG at the C-terminal. Cells were co-transfected with the pcDNA3.1 empty vector, wt-H-Ras, H-Ras^G12V or H-Ras^S17N. Biotinylated membrane ENaC was isolated and immunoblotted with an antibody directed against HA, allowing detection of the 95 kDa un-cleaved and 30 kDa cleaved α-ENaC. C, lower panel, Immunoblot analysis of α-ENaC in total cell lysate. D, upper panel, Immunoblot analysis of membrane γ-ENaC in HEK293 cells transfected with HA (N-terminus) and V5 (C-terminus) tagged γ-ENaC (_HAγ_V5) and α- and β-ENaC with FLAG tagged at the C-terminal. D, lower panel, Immunoblot analysis of γ-ENaC in total cell lysate.