Figure 2: Glycosylation of PD-L1 stabilizes PD-L1 protein.
From: Glycosylation and stabilization of programmed death ligand-1 suppresses T-cell activity
(a,b) Western blot analysis of PD-L1 protein in PD-L1-Flag expressing MDA-MB-231 cells (a) and HEK 293T cells (b). Cells were treated with 20 μM cycloheximide (CHX) at indicated intervals and analysed by western blot analysis. The intensity of PD-L1 protein was quantified using a densitometer. (c) Inhibition of PD-L1 glycosylation enhances ubiquitination. Breast cancer cells with TM and/or MG132 treatment were subjected to PD-L1 immunoprecipitation (IP) and western blot analyses with anti-K48 ubiquitin. (d) Schematic diagram of various PD-L1 NQ mutants used in this study. The numbers indicate amino acid positions on the PD-L1. (e) Protein half-life of PD-L1 WT or various NQ mutants expressing in MDA-MB-231 cells. Experiments were performed as described in (a). Quantification of PD-L1 half-life was shown in (f). (g) Ubiquitination of PD-L1 proteins in PD-L1 WT or various NQ mutants expressing MDA-MB-231 cells. PD-L1 proteins were IP with Flag antibody and then immunoblotted with ubiquitin antibody. Approximately 5% of the cell extract from IP was saved as input. Black circle, glycosylated PD-L1; arrowhead, non-glycosylated PD-L1. All error bars are expressed as mean±s.d. of 3 independent experiments.
