Anti hif1a

Total cellular proteins were extracted from CD4+T cells and spleen mononuclear cells using a protein extraction kit (Keygen Biotech, Nanjing, China). Proteins were separated by 10% SDS/PAGE and transferred to PVDF membranes (Merck Millipore, USA). Membranes were washed three times with TBST, then blocked in TBST containing 5% BSA for 2 h at room temperature. Membranes were then incubated overnight at 4°C with anti-Sirt2, anti-HIF1a, anti-mTOR, anti-LDHA, anti-Glut1, anti-HK2, anti-PKM2 antibody (1:1000 dilution; Cell Signaling Technology) and anti-GAPDH(1:1000; Santa Cruz). Membranes were washed in TBST three times, and then incubated with goat anti-Rabbit IgG secondary antibody (1:5000 dilution; Santa Cruz) solution for 2 h at room temperature. Finally, protein bands were visualized using a chemiluminescence western blot detection system (Alpha Innotech; MicroChemi 4.2).

Protein was extracted from harvested cells using an RIPA buffer (Cell Signaling, #9806) supplemented with PMSF (Cell Signaling, #8553) and protein concentration was calculated with Bradford assay. Total protein extracts were subjected to electrophoresis on 10% SDS-polyacrylamide gels and transferred on PVDF membranes. After blocking with 5% non-fat milk in TBS-T, the membranes were probed overnight at 4 °C with the following antibodies diluted in the same blocking buffer: anti-p53 (1:2000, mouse, Dako, #M7001), anti-HIF-1a (1:1000, rabbit, Cell Signaling Technology, #36169), anti-PD-L1 (1:500 mouse, Origene, #TA808771), and anti-β-actin (1:2000, mouse, Cell Signaling Technology, #3700). Then, the membranes were washed with TBS-T and incubated for 1 h at room temperature with the following species-specific HRP-linked secondary antibodies: anti-mouse (1:4000 Cell Signaling Technology, #7076) and anti-rabbit (1: 4000 Cell Signaling Technology, #7074). After brief washing with TBS-T, membranes were incubated with LumiGLO chemiluminescent substrate and hydrogen peroxide (Cell Signaling Technology, #7003) and pictures were captured using the Invitrogen iBright FL1500 Imaging System.