Protein Extraction and Western Blot Analysis

Phosphorylated and total proteins derived from cells and lung homogenates were lysed in RIPA buffer. The mitochondrial protein was extracted from AMs cells using the Cytoplasmic and Mitochondrial Protein Extraction Kit (Sangon Biotech, Shanghai, China). The samples were separated by electrophoresis on 12% SDS-PAGE gels and transferred to nitrocellulose transfer membranes (Merck Millipore, CORK, IRL). Proteins were detected using primary Abs at a concentration of 1/500 or 1/1000 (all obtained from Cell Signaling Technology, Boston, MA, except IL-1β, β-actin, Affinity Biosciences, Changzhou, China) and incubated overnight. Specific interaction with the primary antibodies was detected using corresponding secondary Abs conjugated to HRP (Biosharp, Hefei, China), and signals were developed using the enhanced chemiluminescence reagents (Biosharp, Hefei, China). UVP ChemStudio (Analytik Jena, Upland, CA, USA) was used for signal detection. Gel bands were quantified by Image J software, and data were presented as means ± SD from three independent immunoblotting assays. Phosphorylated, mitochondrial, and total protein levels were determined and quantified by three successive immunoblotting membranes [63 (link)].