Western blot analysis of cleaved caspase-3 protein [19]

Equal amounts of proteins from lung homogenates were heat-denatured in Laemmli sample buffer with 2-mercaptoethanol (5%), resolved in 15% SDS-PAGE gel and transferred to nitrocellulose membranes (Thermoscientific, Rockford, IL, USA). Next, blots were blocked with 5% PBS-nonfat dry milk for 1 h at room temperature and then incubated with a rabbit monoclonal anti-active caspase-3 (cleaved) primary detection antibody (1:1000) (9661 Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. After thoroughly washing with PBS 0.05% Tween-20, membranes were incubated for 2 h at room temperature with a goat anti-rabbit HRP-conjugated secondary antibody (1:5000) (Thermo Scientific, Rockford, IL). Cleaved caspase-3 immunoreactivity was visualized by enhanced chemiluminescence (SuperSignal™ Pico Chemiluminescent Substrate kit; Thermo Scientific, Rockford, IL, USA). C-DiGit Blot Scanner (Li-Cor, Lincoln, NE, USA) was used to image chemiluminescent signals by scanning. Densitometric analysis was performed using the ImageJ software version 1.46 m (NIH, Bethesda, MD). β-tubulin was used to control for equal loading.