Glutaminase

The total protein was extracted by SDS buffer, and then the protein concentrations were measured using a bicinchoninic acid (BCA) kit (Thermo Fisher Scientific). Subsequently, the proteins (20 µg) were separated by 10% SDS-PAGE, followed by transfer to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk, the membranes were incubated with the primary antibodies at 4°C overnight, using antibodies for PD-L1 (Cell Signaling Technology, Danvers, MA, USA, no 13684, at a dilution of 1:1,000), glutaminase (Abcam, Cambridge, UK, cat. no ab156876; dilution 1:1,000), β-actin (Cell Signaling Technology, no 4970; dilution 1:1,000), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Sigma-Aldrich, no G9545; dilution 1:5,000). Then, the membranes were incubated with HRP-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA; dilution 1:10,000) for another 1 hour at room temperature. The immune complexes were detected using an enhanced chemiluminescence kit (Millipore). The results were normalized to GAPDH or β-actin to correct for the differences in the loading of the proteins. Densitometric analysis was conducted using AlphaView SA software.