Quantification of Lung Protein Markers

Lung tissue from mice was ground into homogenates at the condition of 4°C for protein detection [26 (link)]. Total protein was extracted via a Total Protein Extraction Kit (Invent Biotechnologies Inc., USA) according to the manufacturer's protocol. Enhanced BCA Protein Assay Kit (Beyotime, Jiangsu, China) was used for concentration detection. Protein samples were then separated by SDS-PAGE gel electrophoresis on 12% glycine-based gels and transferred to nitrocellulose membranes (Millipore Corp., Billerica, MA, USA). Membranes were blocked in 5% (w/v) skimmed milk for 2 h and incubated with antibodies in 5% (w/v) BSA overnight at 4°C. Anti-Foxp3 antibody, anti-RORγt antibody, and anti-GAPDH antibody were purchased from Abcam (United Kingdom). Membranes were then incubated with secondary HRP-labeled antibody (Sigma-Aldrich, St. Louis, MO, USA) in 5% (w/v) BSA for 1 h. Afterwards, enhanced chemiluminescence (Beyotime, Jiangsu, China) was developed on membranes. The intensity of bands was quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, USA).