Western blot analysis for Bax, Bcl-2 and cleaved caspase 3 in vivo

The lung tissues were homogenized and analyzed by western blotting. Protein concentrations were determined using the BCA method. An equal quantity of protein was loaded onto 10% sodium dodecyl sulphate-polyacrylamide gels for electrophoresis. Following electrophoresis, the proteins were electroblotted onto polyvinylidene difluoride membranes. Membranes were blocked with blocking solution (5% skimmed milk diluted with PBS) at room temperature for 2 h, followed by incubation with primary antibodies against β-actin (1:5000 dilution; cat. no. ab8227; Abcam, Cambridge, UK), Bax (1:1,000 dilution; cat. no. ab32503; Abcam), Bcl-2 (1:1,000 dilution; cat. no. ab59348; Abcam) and cleaved caspase 3 (1:1,000 dilution; cat. no. ab13847; Abcam) overnight at 4°C. The membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibody (1:5,000, cat. no. ab6721, Abcam), and the protein expression was detected using an enhanced chemiluminescence reagent [cat. no. abs920B-500, Absin Biotechnology Co., Ltd., Shanghai, China].