Western blotting

The right upper lobe of the lungs of the rats in the experimental groups and the control groups was lysed with RIPA buffer (10 mM Tris-HCl, pH 7.2, 0.1% SDS, 1% sodium deoxycholate, 0.15 M NaCl, 1% NP-40), and the lysates were centrifuged at 1,500 x g for in 4˚C for 20 min. The concentration of total protein was determined by the BCA method. The protein concentration was measured by enzyme standard instrument at 562 nm after 30 min. Then, 10% SDS-PAGE gel electrophoresis was performed to isolate and transfer the protein to the PVDF membrane, and the membrane was sealed at room temperature for 1 h with 5% skimmed milk powder. Subsequently, the primary antibodies were incubated with the membrane overnight at 4˚C, and the secondary antibodies were incubated with protein for 1 h at 4˚C. Finally, the exposure and data analysis of protein bands were carried out. Primary antibodies, including anti-p-ERK (cat. no. ab79483; dilution, 1:1,000), p-p38 (cat. no. ab4822, dilution, 1:1,000), p38 (cat. no. ab31828; dilution, 1:1,000), ERK (cat. no. ab17942, dilution, 1:1,000), p-JNK (cat. no. #81E11; dilution, 1:1,000), JNK (cat. no. ab124956, dilution, 1:10,000), SOCS3 (cat. no. ab16030; concentration, 1) and TRAF1 (cat. no. ab129279; concentration, 1 µg/ml), as well as the secondary antibodies, goat anti-rabbit IgG H&L (HRP) (ab6721; dilution, 1:20,000); goat anti-mouse IgG H&L (HRP) (ab6789; dilution, 1:10,000), donkey anti-goat IgG H&L (HRP) (cat. no. ab6885, concentration, 1 µg/ml) were purchased from Abcam. Actin was used as the internal control. Three independent experiments were conducted in each state of rats.