Enhanced chemiluminescence

Total proteins were extracted from lung tissues, primary fibroblasts, and Mlg cells using cold RIPA buffer containing protease and phosphatase inhibitor cocktail (Proteintech, Wuhan, China). The proteins were then separated with 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked in 5% (w/v) non-fat skimmed milk/TBST for 1.5 h. Primary antibodies against α-SMA (1:500, Servicebio), Fibronectin (1:1000, Cell Signaling Technology, Danvers, MA, USA), Collagen-1 (1:1000, Cell Signaling Technology), Grp78 (1:1000, Cell Signaling Technology), CHOP (1:1000, Cell Signaling Technology), p-STAT3 (1:500, Affinity), p-JAK1 (1:1000, Cell Signaling Technology), STAT3 (1:1000, Proteintech), JAK1 (1:1000, Proteintech), VDR (1:1000, Proteintech), β-actin (1:3000, Sigma, St. Louis, MO, USA) and ATF6 (1:500, Affinity, Changzhou, China) were incubated with the membranes overnight at 4 °C. After washing three times with TBST buffer, the membranes were incubated in horseradish peroxidase-labeled secondary antibodies (1:1000, Proteintech) for 1 h at room temperature. The Western blots were subsequently detected using enhanced chemiluminescence (Biosharp, Hefei, China), and the chemiluminescent signals were quantified using Tanon Chemilumenescence Instrument (Tanon, Shanghai, China). The images were analyzed using Fiji software.

Levels of iNOS, Arg1, N protein, and GAPDH were measured by Western blotting. Briefly, treated cells were collected and lysed on ice for 30 min in a protein isolation buffer, subjected to SDS-PAGE, and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% low-fat milk at 37 °C for 1 h and then probed with MAb anti-N (1:100; prepared in the College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China), rabbit anti-iNOS (1:2000, Proteintech), rabbit anti-Arg1 (1:1000; ProteinTech, Rosemont, IL, USA), and rabbit anti-GAPDH (1:1000; Beyotime Biotechnology, Shanghai, China) at 37 °C for 2 h. After washing, the membrane was incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibody (1:10,000, ABclonal). Bound proteins were detected by enhanced chemiluminescence (Biosharp, Hefei, China) and imaged using a chemiluminescence imaging system (Tanon, Shanghai, China).

Cells were lysed in lysis buffer containing phosphatase inhibitors and the concentrations of protein were obtained using bicinchoninic acid (BCA) method (Fdbio science, China). A total of 18 μg of protein was separated by using 10% SDS–PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Ireland). After blocking for 1–2 h, the membrane was incubated with primary antibody anti-MCM3 (ab-DF6205, Affinity Biosciences) and anti-B-actin (20536-1-AP, Proteintech Group Inc.) diluted in 5% BSA or skimmed milk in TBST with shaking overnight at 4 °C followed by HRP conjugated secondary antibody at room temperature for 2 h. B-actin was used as the loading control. The protein expression was detected by using enhanced chemiluminescence (Biosharp, China).