Association of Nedd4-2 with α-ENaC, β- ENaC and γ-ENaC. (A) Protein from alveolar epithelial type II cells lysate was immunoprecipitated with rabbit anti- α-ENaC, β-ENaC and γ-ENaC antibodies or with goat anti-rabbit IgG. Levels of coimmunoprecipitated Nedd4-2 were detected by Western blotting (WB) with an anti-Nedd4-2 antibody. The IgG did not immunoprecipitate Nedd4-2, thus serving as a negative control.(B) Effects of insulin, LY294002 and Akt inhibitor on Nedd4-2 in coimmunoprecipitated α-ENaC, β- ENaC and γ-ENaC. Levels of coimmunoprecipitated Nedd4-2 were significantly decreased by insulin treatment. LY294002 and Akt inhibitor significantly blocked the decrease in coimmunoprecipitated Nedd4-2 induced by insulin. Data are normalized for the amount of Nedd4-2 immunoprecipitated and expressed as fold of the control. Data are presented as mean ± S.E.M.ΔP < 0.05 vs Control group;*P < 0.05 vs Insulin group.