Anti ido1

Total proteins were extracted from OSCC tissues and cells using RIPA lysis buffer. Protein concentrations were determined using BCA protein assay kits (Sigma-Aldrich Corp.). Equal amounts of protein were resolved via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Non-specific protein binding was blocked with non-fat milk in PBS, and then, the membranes were probed at 4 °C for 14–16 h with anti-SRF, anti-E-cadherin, anti-N-cadherin, anti-IDO1, and anti-AhR polyclonal antibodies (all from Proteintech Group Inc., Rosemont, IL, USA), as well as an anti-GAPDH primary antibody diluted 1:2000 (Goodhere Biotechnology, Hangzhou, China). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Biosharp, Hefei, China) for 2 h at 25 °C. The density of immunoreactive bands was visualized using enhanced chemiluminescence (Advansta, San Jose, CA, USA), which was quantified using Image J version 1.48 software (National Institutes of Health, Bethesda, MD, USA).

PC cells were lysed using the RIPA lysis buffer (Beyotime, China; Cat number: P0013B), and supernatants were collected as total protein. The PM fraction of cells was isolated following the standard procedure of membrane and cytosol protein extraction kit (Beyotime; Cat number: P0033). The protein concentration was assessed using the BCA Protein Assay Reagent (Beyotime; Cat number: P0012S). Lysates were subjected to 10% SDS‐PAGE and transferred onto PVDF membranes. Following blocking with PBS containing 5% nonfat milk, membranes were incubated with the respective primary antibodies: anti‐IDO1 (1:5000; Proteintech, USA; Cat number: 66528‐1‐Ig), anti‐HK2 (1:1000; Servicebio, China; Cat number: GB111063), anti‐LDHA (1:2000; Servicebio; Cat number: GB11342), anti‐GLUT1 (1:1000; Servicebio; Cat number: GB113495), anti‐Na+/K+ ATPase (1:1000; Beyotime; Cat number: AF1864), anti‐Bax (1:800; Servicebio; Cat number: GB114122), anti‐Bcl‐2 (1:1000; ABclonal, China; Cat number: A19693), or anti‐β‐actin (1:5000; Abways Technology, China; Cat number: AB2001) antibodies. After incubation with an HRP‐conjugated anti‐mouse secondary antibody (Beyotime) or anti‐rabbit secondary antibody (Beyotime), the proteins were detected using ECL reagents (Thermo Fisher Scientific, USA) and immunoreactive signals were quantitatively assessed using densitometry.