Western blot analysis.

Western blots were generated from mouse lungs as previously described (49). The left lobe of the lung was flash-frozen in liquid nitrogen and homogenized in lysis buffer containing protease inhibitors. Protein concentration was determined using the Pierce bicinchoninic acid protein assay kit (Thermo Scientific). Proteins were separated by size on 6% SDS-polyacrylamide gels and transferred to nitrocellulose. Membranes were blocked with 10% nonfat dry milk in 0.05% Tris-buffered saline + Tween 20 (TBS-T) for 1 h at room temperature and incubated overnight at 4°C with rabbit anti-Atm (clone D2E2, Cell Signaling Technologies, Danvers, MA) or rabbit anti-actin (Sigma) in 5% nonfat dry milk in 0.05% TBS-T. Blots were washed extensively with TBS-T before incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Thereafter, blots were washed extensively, and immune complexes were visualized by chemiluminescence using Pierce ECL Plus Western blotting substrate (Thermo Scientific) and visualized by exposure to BioBlot BXR film (Laboratory Product Sales, Rochester, NY). Exposed film was scanned, and brightness of the blots was adjusted using ImageJ.