As previously described, ubiquitination assay was executed to monitor the ubiquitination of PD-L1 protein.21 22 (link) Briefly, the transfected MDA MB 231 cells and Hs578T cells were harvested and lysed with the RIPA lysis buffer supplemented with protease inhibitor and phosphatase inhibitor (Beyotime, Shanghai, China) for 1 hour at 4°C. Then, the cell lysate samples were incubated with specific antibody for 2 hours at 4°C, with IgG as the control. The beads were subsequently added into the mixture and rotated for 12 hours at 4°C. Through centrifugation, the immunoprecipitated complexes were harvested and boiled in the sodium dodecyl sulfate (SDS) loading buffer for 5 min. Later, WB assay was carried out to detect the ubiquitination of PD-L1 protein.