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. 2020 Nov 16;11(3):694–707. doi: 10.1016/j.apsb.2020.11.005

Figure 3.

Figure 3

Abrogation of USP7 downregulated PD-L1 expression by increasing PD-L1 polyubiquitination. (A) mRNA levels of PD-L1 when MGC-803 cells were treated with different concentrations of Almac4 or 5 μmol/L Almac4 for different times; the values were normalized on the PD-L1 expression in control samples. (B) Interaction analysis between Flag-PD-L1 and HA-USP7 using IP. HEK293 cells were transiently transfected with the indicated plasmids Flag-PD-L1 (left) and HA-USP7 (right) was immunoprecipitated and subjected to WB analysis with the HA-tag (left) and Flag-tag antibody (right). (C) Endogenous PD-L1 IP in MGC-803 and BGC-823 cells. (D) WB analysis of Flag-PD-L1 expression in HEK293 cells transfected with Flag-PD-L1 and dose increasing amounts of HA-USP7. (E) Polyubiquitination of PD-L1 in HEK293 cells when Flag-PD-L1 and HA-His-ubiquitin were co-transfected with HA-USP7 or not; cells were treated with MG132 (10 μmol/L) or not 6 h prior to ubiquitination analysis. (F) Polyubiquitination of PD-L1 in MGC-803 cells when USP7 was abrogated genetically, cells were treated with MG132 (10 μmol/L) or not 6 h prior to ubiquitination analysis. Turnover of PD-L1 in MGC-803 cells when USP7 was abrogated genetically (G) or pharmacologically (H) with 20 μmol/L CHX for different times as indicated. Data are shown as mean ± SD (n = 3); ns, not significant; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. P values were calculated using two-tailed t-test statistical analysis.