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. 2020 Dec 16;28(6):1773–1789. doi: 10.1038/s41418-020-00700-z

Fig. 3. OTUB1 stabilizes PD-L1 through cleaving K48-linked poly-ubiquitin chains of PD-L1.

Fig. 3

a HA-PD-L1 ubiquitination levels in HEK293T cells transfected with Flag-vector or Flag-OTUB1 were analyzed by His pull-down assay. b The effect of PD-L1 ubiquitination by depleted OTUB1 was examined in OTUB1WT, OTUB1-/- and re-expressed OTUB1 HEK293T cells by His pull-down assay. Two OTUB1-/- strains were obtained through CRISPR/Cas9 genome editing. c The shControl- and shOTUB1- MDA-MB-231 cells were subjected to TUBE pull-down assays to examine the effect of depleted OTUB1 on endogenous ubiquitination levels of PD-L1. d The influence of OTUB1 on specific ubiquitination types of endogenous PD-L1 was determined by immunoblotting analysis using the K48- or K63- ubiquitin linkage-specific antibodies. MDA-MB-231 cells infected with shControl- or shOTUB1-encoding lentiviruses were treated with MG132 overnight and then subjected to immunoprecipitation. e In vitro deubiquitination assays of recombinant OTUB1 proteins and enriched K48-linked or K63-linked ubiquitinated PD-L1 from cell extracts. The mixture was incubated at 30 °C for 4 h and then analyzed by immunoblotting. f Schematic representation of the OTUB1 C91S, D88A, ASA (D88A/C91S/H265A) mutants and ΔN truncation. Stars represent mutated amino acids in the OTU domain. g Examination of the effect of wild-type OTUB1 or ASA mutant on PD-L1 abundance. GFP expression was used to confirm constant transfection efficiency across the three experimental groups. h Determination of PD-L1 levels after re-expression of the indicated OTUB1 mutants. The shOTUB1 MDA-MB-231 cells were infected with lentiviruses stably expressing shRNA-resistant wild-type OTUB1 or ASA mutant and then subjected to immunoblotting of PD-L1. i Immunoblot analysis of PD-L1 ubiquitination affected by wild-type OTUB1, ASA, C91S, D88A, or ΔN truncation mutants. HEK293T cells were transfected with HA-tagged PD-L1 and His-ubiquitin along with different OTUB1 mutants. Following MG132 treatment, cell extracts were enriched by Ni+-NTA agarose and then prepared for immunoblotting with the indicated antibodies. j Detection of endogenous K48-linked polyubiquitination of PD-L1 affected by OTUB1. MDA-MB-231 cells with shControl or shOTUB1 and OTUB1-depleted cells with re-overexpressed wild-type or mutation constructs of Flag-OTUB1 were subjected to K48-TUBE pull-down analyses.