Tgf β1

Frozen jejunum and colon tissue samples were homogenized in RIPA lysis buffer containing protease inhibitor. The total protein concentration was determined using a BCA protein assay Kit (Beyotime Biotechnology, Shanghai, China). Approximately 30 mg of protein were loaded and separated by SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skimmed milk for 2 h at room temperature and then incubated with primary antibodies: PCNA, EGFR, and TGF-β1 (1:750) (Wanlei Biotechnology, Liaoning, China) overnight at 4°C, and then incubated with secondary antibody for 1 h at room temperature. The expression of target proteins was detected with ECL chemiluminescence reagents (E412-01; Vazyme Biotechnology, Nanjing, China) and Imager-Bio-Rad (Bio-Rad Laboratories, Inc., Hercules, CA, United States), then quantified using Image J software.

Recombinant human IL-1β was purchased from Sigma-Aldrich (#SRP3083, St. Louis, MO, USA). The GV248 lentiviral short-hairpin (sh) RNA-expression vector targeting human NF-κB (p65; shp65), human TIR8 (shTIR8), scrambled shRNA (shSc), and the GV248 lentiviral vector were obtained from Genechem (Shanghai, China). The target sequences are listed in Supplementary Table 1. Packaging vectors (pVSVG, pREV, and pMDL) were used as described previously (Zha et al. 2015 (link)). Human TIR8 cDNA was subcloned at BamHI and EcoRI sites of the pcDNA3.1-C/DYK expression vector.
Primary antibodies used for western blotting analysis include, TIR8 (Abcam, #ab228977. Cambridge, MA, USA), E-cadherin (CST, #14,472. Shanghai, China), vimentin (CST, #5741), p65 (CST, #8242s), phosphorylated p65 (p-p65 at Ser536. CST, #3031s), TGF-β1 (Proteintech, #21898-1-AP. Rocky Hill, NJ, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Image Bioscience, #IMA1004L. Beijing, China). Primary antibodies against E-cadherin (CST, #14,472), vimentin (CST, #5741), p-p65 (Santa Cruz, #SC136548. CA, USA), TIR8 (Abcam, #ab228977), IL-1β (Wanleibio, #WL00891. Shenyang, China), and TGF-β1 (Wanleibio, #WL02193) were used to stain the corresponding proteins, and IgG (CST, #4414S) was used as a negative control.

Lung tissues and PASMCs were lysed in radio immunoprecipitation assay (RIPA) lysis buffer (containing 1% PMSF) on ice to extract total proteins. Nuclear and cytoplasmic proteins were extracted following the manufacturer's recommendations of the nuclear and cytoplasmic extraction kit (Beyotime). After protein quantification, the same amount of protein from each sample were separated on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and then the proteins on the gel were transferred to PVDF membranes (Millipore) by semi‐dry electrophoretic transfer system (Bio‐rad). The membranes were then blocked with 5% BSA and incubated with primary antibodies against calpain‐1 (1:1000, proteintech), HIF‐1α (1:1500, ABclonal), P65 (1:2000, proteintech), β‐actin (1:5000, proteintech), Lamin B (1:10000, proteintech), VEGF (1:1000, ABclonal), TGF‐β1 (1:800, Wanleibio), PCNA (1:1500, ABclonal), MMP2 (1:1000, ABclonal) and collagen I (1:1000, Wanleibio) overnight at 4°C. On the second day, membranes were incubated with HRP‐conjugated secondary antibodies (1:10000, ABclonal). The immune response bands were visualized with a chemiluminescence reagents (Biosharp).