Protease inhibitor

Total ovarian proteins were extracted by using RIPA lysis buffer containing protease inhibitors and protein phosphatase inhibitors (Servicebio Biotechnology Co., Ltd., Wuhan, China). The mixtures were homogenized and stored on ice for 10min, and then centrifuged at 12,000 rpm for 10 min at 4° C. The supernatant was used to conduct western blot analysis. The proteins (30 μg) were separated with 10% SDS-polyacrylamide gels and then transferred onto PVDF membranes (Millipore, Burlington, USA). After being blocked with 5% skimmed milk for 1 h, the strips of target proteins and internal protein were washed with TBST. The strips were each incubated with primary antibodies against TGF-β1, p-Smad3, Smad3, Smad7, MMP2, and GAPDH purchased from Servicebio Biotechnology Company (Wuhan, China) overnight at 4° C. The strips were then incubated with HRP-conjugated anti-rabbit IgG (CST, Danvers, USA) secondary antibodies for 2h at room temperature. The contents of target proteins were detected with an enhanced chemiluminescence agent (Tanon, Shanghai, China). The grey values of the protein strips were analyzed with Image J software (National Institutes of Health, Bethesda, USA). The expressions of target proteins were normalized to the protein of GAPDH.

Cells and tissues were lysed in RIPA buffer (Servicebio, Wuhan, China) with protease inhibitors (Servicebio, Wuhan, China). Total proteins were extracted and separated by 10% SDS-PAGE, then transferred to PVDF membranes. Membranes were blocked with 5% skim milk (BD, USA) for 1 h at room temperature before incubating with primary and secondary antibodies. Protein bands were visualized using ECL reagent (Servicebio, Wuhan, China). Antibody information is listed in Supplementary Table S1.

Tissues were grinded in a homogenizer (KZ-III, Servicebio, China) and proteins were isolated with RIPA lysis buffer (Beyotime Biotech, China) supplemented with protease inhibitors (Servicebio, China). After quantification, the proteins were subjected to SDS-PAGE electrophoresis and immediately transferred to a polyvinylidene fluoride membrane (Millipore, CA, USA). the blots were blocked with 5% skim milk and then incubated with primary antibodies at 4°C overnight. The primary antibodies used for this were: anti-α-SMA (Proteintech 14395-1-AP), anti-Collagen I (Abcam ab6308), anti-Fibronectin (Proteintech 15613-1-AP), anti-β-Tubulin (Fude technology FD0064) and anti-β-Actin (Proteintech 66009-1-Ig). Next, goat anti-rabbit or anti-mouse antibodies (Biosharp Biotech; 1:10000) were then applied, and immunoreactivity was detected using enhanced chemiluminescence (Fude technology, China) and imaged with Chemiluminescence Imager system. Quantitative analysis was performed via Image J software.

HT29 and CT26.WT were cultured in a six-well plate for 48 h; total protein was extracted using RIPA buffer (Servicebio, G2002, China) containing protease inhibitors (Servicebio, G2006, China) and quantified using BCA kit (Thermo Fisher Scientific, USA). For each sample, 25 μg total protein was separated by 10% SDS-PAGE and transferred to a PVDF membrane using a wet transfer blotting system (BioRad, Hercules, CA), antibodies such as anti-IRF-1 (Cell Signaling Technology, #8474, USA), anti-IRF-2 (Abcam, ab1274744, USA), anti-E-cadherin (ABclonal, A3044, China), anti-N-cadherin (ABclonal, A0433, China), anti-Vim (ABclonal, A11423, China) were used for western blotting; anti-histone (Abcam, USA) was used as an endogenous control.

Total and nuclear proteins were extracted from BMDMs in RIPA buffer containing protease inhibitors (Servicebio, Wuhan, China). Then protein extracts were separated by 10% SDS-PAGE and incubated overnight at 4°C with primary antibodies specific for AhR (BA2013, 96 kDa, 1:200, Boster, China), HIF-1α (PB9253, 97 kDa, 1:1,000, Boster, China), IRF1 (abs118047, 37 kDa, 1:1,000, Absin, China), NF-κB p65 (GB11142-1, 65 kDa, 1:1,000, Servicebio, China), iNOS (BA0362, 130 kDa, 1:200, Boster, China), Arg-1 (GB11285, 40 kDa, 1:5,000, Servicebio, China), H3 (GB13488, 15 kDa, 1:1,000, Servicebio, China), and β-actin (BA2305, 43 kDa, 1:1,000, Boster, China). Blottings were then probed with appropriate secondary antibodies for 2 h at 25°C and assessed via an ECL kit (Millipore, MA, USA). Triplicate analyses of all samples were conducted.

The hepatic tissues were lysed with RIPA lysis buffer (Servicebio technology, Wuhan, China), supplemented with protease inhibitor (Servicebio), and homogenized with an ultrasonic processor. According to the manufacturer’s instructions, the total protein of the liver tissue was extracted with a commercial kit (Servicebio technology, Wuhan, China). Then, the concentration of the protein was measured with a BCA kit (Beyotime technology, Shanghai, China). Then, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, followed by blocking with 5% skim milk (0.5% TBST) and sealed for 1 h. Additionally, then, PDVF membranes were incubated with primary antibodies against NF-κB (1:1000), p-ERK (1:1000), PI3K (1:1000), p-AKT (1:1000), Caspase-3 (1:1000), Bcl-2 (1:1000), Bax (1:1000), and GAPDH (1:3000) were incubated overnight at 4 °C. They were washed with TBST at room temperature on a decolorizing shaker three times. After washing, PVDF membranes were incubated with secondary antibodies (1:3000) at room temperature for 2 h. The antibodies were purchased from Proteintech Group, USA. Finally, they were developed and fixed with developing and fixing reagents, and the Alpha software processing system analyzes the optical density values of the target band.