Western blot analysis of rat lung tissues and A549 cells

Proteins were obtained with RIPA lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, and leupeptin) and PMSF. The protein concentrations of the supernatants were determined by using a BCA protein assay kit. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. Tris buffer solution containing 5% skim milk powder was used to block the membrane for 1 h at room temperature. The membrane was permeabilized with PBS containing 0.05% Tween 20 and washed with PBST 5 times (5 min each time). Then, the membrane was incubated overnight at 4°C with the following primary antibodies: α-, β-, and γ-ENaC (Abcam, Cambridge, UK), Akt, p-Akt, Nedd4-2, and β-actin (CST, Boston, MA, USA). The membrane was incubated with a horseradish peroxidase (HRP)-labeled goat anti-rabbit antibody (Abcam, Cambridge, UK) at room temperature for 3 h, and the membrane was washed with PBST 5 times (5 min each time). Finally, the bands were visualized using an enhanced chemiluminescence kit (ECL) with a UVP gel imaging system (Upland, CA, USA). The band intensities were analyzed with ImageJ software