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. 2023 Feb 1;133(3):e160456. doi: 10.1172/JCI160456

Figure 4. MIB2 promotes K63-linked ubiquitination of PD-L1.

Figure 4

(A) Direct binding between PD-L1 and MIB2. FLAG-MIB2 immunoprecipitated from 293T cells bound to purified GST–PD-L1 in vitro. Purified GST–PD-L1 was examined by Coomassie Blue staining. (B) Co-IP analysis of the endogenous PD-L1 and MIB2 in MC38 cells. (C) Proximity ligation assay (PLA) analysis of PD-L1 and MIB2 in A375, B16-F10, and MC38 cells. Scale bar: 10 μm. (D) In vivo PD-L1 ubiquitination by MIB2 in 293T cells transfected with expression constructs. (E) Immunoprecipitation (IP) and immunoblotting (IB) analysis of 293T cells transfected with various ubiquitin mutant constructs. (F) IP and IB analysis of MIB2-KO MC38 cells using Ub-K63–specific antibody. (G) IP and IB analysis of PD-L1 in A375 cells interacting with purified HIS–PD-1.