Ab211017

Total protein extracts of cell lines or tissues were prepared by using radioimmunoprecipitation assay buffer (Sigma-Aldrich) containing protease inhibitors. The protein concentration of lysates was quantified by using a bicinchoninic acid protein assay (Beyotime, China). For Western blotting, 20 μg of the protein sample was separated by electrophoresis on a 10% sodium dodecyl sulfate–polyacrylamide gel and was transferred to a nitrocellulose membrane (Sigma-Aldrich; Merck KGaA). Then, the membrane was blocked with 5% nonfat milk in PBS for 1 hour at room temperature and immunoblotted at 4°C overnight with a primary antibody against one of the following proteins: IDO1 (1:1000, ab211017, Abcam, USA), programmed cell death ligand 1 (PD-L1) (1:1000,ab213524, Abcam), STAT3 (1:1000,ab68153, Abcam), p-STAT3 (1:1000,ab76315, Abcam), E-cadherin (1:5000ab40772, Abcam), N-cadherin (1:5000,ab76011, Abcam,), vimentin (1:5000,ab92547, Abcam), and actin (1:5000,sc- 47778, Santa Cruz Biotechnology, Dallas, TX), which was used as an internal control. Subsequently, the membranes were incubated with fluorescence-conjugated secondary antibody (926-6807 or 926-32210; LI-COR Biosciences, Shanghai, China) for 1 hour at room temperature. The labeled protein bands were visualized and quantified by using the Odyssey two-color infrared laser imaging system (LI-COR Biosciences, Lincoln, NE, USA).

The total proteins were extracted from cells by RIPA lysis buffer (Beyotime) with phenylmethanesulfonyl fluoride (PMSF; Beyotime) and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime) transferring to polyvinylidene difluoride membranes (PVDF; Millipore, MA, USA). Primary antibodies against type I collagen (COL1) (1:1000, Bioss, Beijing, China), runt-related transcription factor-2 (RUNX2) (1:1000, 12556, CST, MA, USA), ALP (1:1000, ab67228, Abcam), NRF2 (1:1000, ab62352, Abcam), NAD(P)H quinone oxidoreductase 1 (NQO1) (1:10,000, ab80588, Abcam), CD206 (1:1000, ab125028, Abcam), iNOS (1:1000, 18985-1-AP, Proteintech, Wuhan, China), IDO (1:1000, ab211017, Abcam), IL-1β (1:1000, A1112, Abclonal), AhR (1:1000, ab190797, Abcam), PCNA (1:500, bs-0754R, Bioss), GAPDH (1:2000, Bioss) were used. The secondary antibody goat anti-rabbit IgG H&L (HRP) (1:5000, ab205718, Abcam) was used. The images were detected by using ChemiDocTM MP Imaging System (Bio-Rad, USA).