Anti cox 1

Cultured cells, isolated tumor cells and stromal cells were lysed with RIPA lysis buffer (Thermo Fisher Scientific, MA, U.S.A) by gently shaking on ice for 30 min. Tumor tissues were homogenized with RIPA lysis buffer. After centrifugation at 12,500 g for 10 min, the supernatants were collected and the concentrations of proteins were measured using Pierce BCA Protein Assay Kit (ThermoFisher Scientific, MA, U.S.A). An equivalent amount of protein was resolved by 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad, CA, U.S.A). The membranes were then blocked in 5% non-fat powdered milk dissolved in phosphate buffer containing 0.05% Tween-20 (PBST) for 1h. Afterwards, the membranes were incubated with anti-COX-1 (Cell Signaling Technology, MA, U.S.A), anti-COX-2 (Cell Signaling Technology, MA, U.S.A) or anti-PD-L1 antibody (Abcam) in antibody dilution buffer (5% BSA in PBST) with gentle agitation overnight at 4 °C. After washing with PBST for three times, the membranes were subsequently incubated with the secondary antibody (Cell Signaling Technology, MA, U.S.A) for 1 h at room temperature. The membranes were then washed three times with PBST before being exposed to the SuperSignal West Dura Extended Duration substrate (Thermo Fisher Scientific, MA, U.S.A). Protein expression was normalized against GAPDH expression.