Anti p stat3

The cells transfected for 48 hours were cleaved using RIRP Lysis Buffer (Thermo Fisher Scientific). Then, the supernatant was collected after centrifugation 4 times at 14 000 rpm for 15 minutes. The protein concentration was detected by bicinchoninic acid (BCA) kit (Bio-Rad, USA). The target protein was separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred into PVDF (polyvinylidene difluoride) membrane overnight. After the blots were sealed using 10% skim milk, the primary antibody was used to hybridize with the target protein overnight at 4°C (anti-MRP1, 1: 1000; anti-GST-π, 1: 2000; anti-ABCB1, 1: 1000; anti-GAPDH, 1: 5000; anti-SOCS2, 1: 1000; anti-SOCS4, 1: 1000; anti-SOCS5, 1: 1000; anti-p-JAK2, 1: 2000; anti-JAK2, 1: 2000; anti-p-STAT3, 1: 2000; anti-STAT3, 1: 2000; Thermo Fisher Scientific). Subsequently, the blots were incubated with horseradish peroxidase conjugated secondary antibody (1: 10 000) after washing in TBST. We used an ECL (enhanced chemiluminescence) kit (Abcam, USA) developed the blots.

To determine the expression of target proteins, the following antibodies were used. Anti-CD163 (Abcam, Cambridge, MA, USA, ab182422), anti-CD80 (ThermoFisher Sci., MA5–15512), anti-iNOS (ThermoFisher Sci., PA1–036); anti-Stat1 (Cell Signaling, 9172 T), anti-p-Stat1 (Tyr701, Cell Signaling, 7649 T), anti-STAT3 (Cell Signaling, 9139 T), anti-p-Stat3 (Ser727, ThermoFisher Sci., 44–384G), anti-IL-12 (R&D, AF309-SP), anti-IL-10 (R&D, AF217-SP), anti-p-Smad3 (Novus Bio, Centennial, CO, USA, nbp1–77836), anti-PD-L1 (Abcam, ab205921, for western blot), anti-PD-L1 polyclonal antibody (Biorbyt, LLC, San Francisco, CA, USA, orb74809, for IHC), anti-β-Actin (Sigma-Aldrich, A1978); anti-mouse CK 8/18 (DSHB, Iowa City, IA, USA, Troma-I); anti-mouse CK 14 (BioLegend, San Diego, CA, USA, 905301); and anti-mouse F4/80 (eBioscience, Waltham, MA, USA, BM8).