Western Blot Analysis of PD-L1 in Tumor Tissue

The tumor tissue (10 mg) was boiled in a Western blotting (WB) sample buffer for 30 min. The proteins in the WB samples were separated by gel electrophoresis (80 V for 30 min followed by 120 V for 1 h) and transferred to a PVDF membrane (100 V for 40 min). The proteins were incubated with a PD-L1 monoclonal antibody (Cat# 66248–1-Ig, ProteinTec Chicago, IL, USA) [23 (link)] or β-actin polyclonal antibody (Cat# 20536-1-AP, ProteinTech Chicago, IL, USA) [24 (link)] and secondary antibody (Cat# SA00001-1, ProteinTech Chicago, IL, USA) [25 (link)], and the expressions of the mouse PD-L1 and actin were analyzed using a ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).