Anti ido

Total protein samples from T cells or DCs were obtained in RIPA lysis buffer followed by centrifugation (12,000 rpm and 10 min) in 4°C. Then, the protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific). Samples were run on 10% SDS-PAGE gels and electro-transferred onto a PVDF membrane. TBST with 5% (w/v) BSA was used to block non-specific binding to the membrane at room temperature for 1 h. The membrane was then incubated with a primary antibody anti-phospho-P70S6K, anti-P70S6K, anti-phospho-mTOR, anti-mTOR (1:1,000; Cell Signaling Technology, USA), or anti-IDO (1:1,000; Millipore, USA) at 4°C overnight. After incubation, the membrane was washed and incubated with a secondary antibody, HRP-conjugated goat anti-rabbit IgG (1:2,000) for 1 h. GAPDH (1:2,000; Cell Signaling Technology) was also used for loading controls. Finally, blots were visualized by an ECL method (Beyotime, China) and analyzed using an Image J Program software.

Tissues or cells were lysed, supernatants were collected and protein concentration was determined with the BCA Protein Assay Reagent (Thermo Scientific, Waltham, MA). Lysates (30 μg for cells, 80 μg for tissues) were separated by 10% SDS-PAGE and transferred electrophoretically onto polyvinylidene difluoride membranes. After blocked with TBST containing 5% non-fat milk, membranes were incubated with the following primary antibodies: anti-IDO (1:500, Millipore, Billerica, MA, No. MAB5412), anti-pSTAT3 (1:2000, Cell Signaling Technology, Beverly, MA, No. 4113), anti-STAT3 (1:2000, Cell Signaling Technology, No. 4904), anti-MMP9 (1:1000, Cell Signaling Technology, No. 3852), and anti-GAPDH (1:5000, Kangchen, No. KC-5G4). After incubation with an HRP-conjugated anti-mouse secondary antibody (1:2000, Cell Signaling Technology, No. 7076) or anti-rabbit secondary antibody (1:2000, Cell Signaling Technology, No. 7074), products were visualized with ECL reagents (Thermo Scientific) and immunoreactive signals were analyzed by densitometry. The intensity of each target protein band was quantified by densitometry analysis using Image J software (version 1.46r, National Institutes of Health, Bethesda, MD). GAPDH was used as an endogenous loading control.