Protease and phosphatase inhibitors

Total proteins were extracted from lung tissues using a radioimmunoprecipitation assay buffer (Beyotime, China) supplemented with protease and phosphatase inhibitors (Biosharp, China). Their levels were quantified using a bicinchoninic acid kit. Proteins were separated with sodium dodecyl sulfate–polyacrylamide gel and transferred to polyvinylidene fluoride membranes. They were blocked in 5% skim milk at 37 °C for 1 hour and incubated overnight at 4°C with primary antibodies: anti-PTEN (1:2000; Abcam, USA), anti-AKT (1:4000; Proteintech, China), anti-p-AKT (1:4000; Proteintech, China), anti-Bcl-2 (1:1000; Proteintech, China), anti-Bax (1:2000; Abcam, USA), anti-Cleaved Caspase 3 (1:2000; CST, USA), anti-SPC (1:1000; Proteintech, China), anti-AQP5 (1:1000; Proteintech, China), anti-Claudin 4 (1:4000; Proteintech, China) and anti-Keratin 8 (1:4000; Proteintech, China). β-actin (1:10000; Proteintech, China) was used as a reference protein. The membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:10000; Proteintech, China) at 37 °C for 1 hour. Protein bands were detected with enhanced chemiluminescence detection reagents (Millipore, USA) and analyzed using ImageJ software (version 1.51; National Institutes of Health, USA).

Peripheral blood mononuclear cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Biosharp, China) containing protease and phosphatase inhibitors (Biosharp, China) to extract total protein. The primary antibodies used in this study included anti-β-actin (1 : 1000, TA-09; Zs-BIO, China), anti-COX2 (1 : 1000, AF7003; Affinity, China), anti-IL-6 (1 : 1000, DF6087; Affinity, China), and anti-TNF-α (1: 1000, AF7014; Affinity, China). The secondary antibodies included goat anti-rabbit IgG (ZB-2301; Zs-BIO, China), anti-β-actin (1: 10000, TA-09; Zs-BIO, China), anti-COX2 (1: 5000, AF7003; Affinity, China), anti-IL-6 (1: 5000, DF6087; Affinity, China), and anti-TNF-α (1: 5000, AF7014; Affinity, China).