Western Blot Analysis of Lung Proteins

Protein samples were prepared from the lung tissues using 1× cell lysis buffer (Cell Signalling Technology, USA). Equivalent amounts of proteins (10–20 µg) were used to assess protein expressions as described previously [11 (link),12 (link)]. Briefly, protein was denatured by boiling, separated by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE, 12%) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 8% non-fat milk/PBS-T (PBS with 0.5% Tween-20) for 2 h and respectively incubated with the following rabbit-derived primary antibodies: NF-κB, Bax, Bcl-xL, cleaved-Caspase-3 and β-actin (all 1:1000 dilution; Sigma Aldrich, St. Louis, MO, USA). After washing, the membranes were probed with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000 dilution; Cell Signalling Technology, USA). Proteins of interest were visualised using the enhanced chemiluminescence kit (EMD Millipore, USA), and the band intensities were quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, USA).