We isolated the cytoplasmic and nuclear fractions from the lung tissue and determined the protein content using a Bradford assay. Subsequently, lung protein lysates (30 μg per lane) were separated via 10%–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblots were then performed in accordance with the method described previously.9 (link)
The blots were subjected to probing with specific antibodies, including anti-ALDH2, anti-4-HNE (diluted at 1:1000, Abcam, Waltham, MA, USA), anti-PI3K, anti-AKT, anti-pAKT, anti-cleaved caspase 3, anti-NF-κB p65, anti-phospho-NF-κB p65, anti-IκB-α (diluted at 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-Bcl-2 (diluted at 1:1000, Bioss Inc., Woburn, Massachusetts, USA), anti-lamin B1 (diluted at 1:200, Santa Cruz Biotechnology, Dallas, TX, USA), or β-actin (diluted at 1:10,000, Sigma Chemical Company, St. Louis, MO, USA). The data were expressed by determining the relative ratio of the target protein's content to that of the reference protein. The relative ratio of the target protein's content in the control group was established as 1.
The blots were subjected to probing with specific antibodies, including anti-ALDH2, anti-4-HNE (diluted at 1:1000, Abcam, Waltham, MA, USA), anti-PI3K, anti-AKT, anti-pAKT, anti-cleaved caspase 3, anti-NF-κB p65, anti-phospho-NF-κB p65, anti-IκB-α (diluted at 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-Bcl-2 (diluted at 1:1000, Bioss Inc., Woburn, Massachusetts, USA), anti-lamin B1 (diluted at 1:200, Santa Cruz Biotechnology, Dallas, TX, USA), or β-actin (diluted at 1:10,000, Sigma Chemical Company, St. Louis, MO, USA). The data were expressed by determining the relative ratio of the target protein's content to that of the reference protein. The relative ratio of the target protein's content in the control group was established as 1.
