Lungs from each group of mice were homogenized in 500 μl lysis buffer (10 mM Tris-HCL pH 7.5, 120 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 1% Triton X-100) containing protease and phosphatase inhibitor cocktails (Roche) using a sonicator on ice. Samples were centrifuged at 14,000 g for 20 min at 4 °C, and the supernatants were collected. Sample protein concentrations were measured using the BCA protein assay kit (Pierce Biotechnology, Rockford, IL), and 65-μg protein samples were separated by electrophoresis on 4–12% gradient Bis-Tris gels and transferred to nitrocellulose membranes. After blocking with 0.1% casein, membranes were incubated with primary antibodies against BiP, pIRE1α, CHOP, cleaved caspase-12 (Cell signaling Technology, Danvers, MA), sXBP1, NF-κ p65 (Santa Cruz Biotechnology, Santa Cruz, CA), 4-HNE (Abcam, Cambridge, MA), or β-actin (Sigma-Aldrich, St. Louis, MO). 4-HNE is stable, making its quantification more reliable than direct quantification of ROS
After washing, membranes were incubated with the appropriate fluorescent-conjugated secondary antibodies. Bands were detected using an Odyssey FC Imaging system (LI-COR, Lincoln, NE). Band intensities were quantified with densitometry and represented as fold changes relative to controls. The corrected band intensity data from two Western blots was used to plot each histogram.
After washing, membranes were incubated with the appropriate fluorescent-conjugated secondary antibodies. Bands were detected using an Odyssey FC Imaging system (LI-COR, Lincoln, NE). Band intensities were quantified with densitometry and represented as fold changes relative to controls. The corrected band intensity data from two Western blots was used to plot each histogram.