Anti cd73

Reagents for SDS-PAGE were obtained from Bio-Rad (Richmond, CA, USA); dihydroethidium (DHE), RPMI 1640 culture medium, heat-inactivated fetal bovine serum, Opti-MEM medium, Lipofectamine 2000, RIPA lysis buffer and Fura-2 AM were obtained from Thermo Fischer Scientific (Waltham, MA, USA); Bradford Reagent and linoleic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA); Insulin Ultra-Sensitive Assay kit was obtained from Cisbio (Codolet, France); GW9508 was obtained from Tocris Bioscience (Bristol, England, UK); VAS2870 was obtained from Enzo Life Sciences (Farmingdale, NY, USA); p22^phox siRNA (#sc-61892), control siRNA (#sc-37007), anti-p47^phox (#sc-14015; dilution 1:1000 in 5% BSA in TBST), anti-p22^phox (#sc-271968; dilution 1:500 in 5% BSA in TBST) and anti-CD73 (#sc-25603; dilution 1:1000 in 5% BSA in TBST) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); goat anti-rabbit IgG HRP (#ab205718; dilution 1:10,000 in 3% skimmed milk in TBST) and goat anti-mouse IgG HRP (#ab97023; dilution 1:10,000 in 3% skimmed milk in TBST) secondary antibodies were obtained from Abcam (Cambridge, UK).

Cells were harvested and lysed with lysis buffer (Beyotime, China) containing protease inhibitors (PMSF, Dingguo, China). Cellular proteins were collected after centrifugation at 10 000 × g at 4°C, and boiled for 5 min together with loading buffer. Proteins were separated by 10% SDS-PAGE, and further transferred onto nitrocellulose membranes (Bio-Rad, USA). The membranes were incubated with primary antibodies (anti-Atg5, Santa Cruz, USA; anti-LC3I/II, Santa Cruz, USA; anti-CD73, Santa Cruz, USA; anti-JAK1, Santa Cruz, USA; anti-STAT3, Santa Cruz, USA; anti-P-JAK1, Cell Signaling Technology, USA; anti-P-STAT3, Cell Signaling Technology, USA) overnight at 4°C after blocking with 5% fat-free milk in TBS-T for 1 h at room temperature. Then the membranes were incubated with a HRP-conjugated secondary antibody for 1h at room temperature followed by three-time washing with TBS-T. Bands were detected with ELL by X-ray films (MidSci, St. Louis, MO, USA) after three-time washing with TBS-T.