Pd l1 e1l3n

Cells were pelleted, washed with PBS, and then lysed with Radio Immunoprecipitation Assay Lysis (RIPA) buffer (Thermo Scientific) supplemented with protease inhibitor cocktail (CST). Protein concentration was determined by the BCA Protein assay kit (Thermo Scientific) and lysates were then boiled for 10 min. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Pall). Membranes were blocked for 1 h at room temperature with 5% milk in 1 × Phosphate Buffered Saline Tween-20 (PBST) and incubated with the indicated primary antibodies at 4 °C overnight. After washing with PBST three times for 30 min, membranes were incubated with secondary antibodies at room temperature for 1 h. The membranes were washed with PBST three times and visualized with chemiluminescence (CLiNX). Antibodies against the following human proteins were used: GAPDH (SCB), PD-L1 (Yurogen), PD-L1 E1L3N (CST), and PD-L1 28-8 (Abcam). Band intensities were quantified by ImageJ and were normalized using GAPDH as a housekeeping protein. Results are representative of three independent experiments.