Fig. 6

ISG15 and PD-L1 form ISGylation and promote its ubiquitination for proteasome-dependent degradation. A Quantitative qRT-PCR analysis of the mRNA level of PD-L1 in Sh-ISG15 and Lv-ISG15 or the control A549/H1299 cells. B Western blotting was performed to detect the expression level of glycosylated PD-L1 at different times in A549 and H1299, after CHX (25 μg/ml) treatment. The data were normalized for comparative purposes (bottom panel). C Ubiquitination level of PD-L1 detected by Immunoprecipitation (IP) assay in Lv-ISG15 or control cells after treated with MG132 (20 μmol) for 8 h. D Immunoprecipitation of ISG15 and PD-L1 at lysine48-linked ubiquitination sites after H1299 and Pc-9 treated with 20 μM MG132 6 h. E Western blot was used to examine the effect of bafilomycin or MG132 on PD-L1 levels in the presence of CHX in Lv-ISG15 cells. F IP experiment detects the interaction between ISG15 and PD-L1. G Co-IP assay detects whether the interaction between ISG15 and PD-L1 is specific to ISGylation and whether it is dissociated by USP18 in H1299 and Pc-9 cells co-transfected ISG15 and USP18 or control plasmids