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. 2020 Oct 6;137(12):1615–1627. doi: 10.1182/blood.2020004918

Figure 4.

Figure 4.

A20 inhibits MYD88L265P K63-linked polyubiquitination and abrogates NF-κB activation. (A) Schematic workflow of DUB screening assay using 293T-NF-κB-Luc cells. (B) Effect of DUB on NF-κB luciferase activity in 293T-NF-κB-Luc cells. Cells were transfected with vector control or cotransfected MYD88L265P /MYD88WT, RNF138, and DUBs and examined for luciferase activity. Blue spot, A20; gray spot, vector control. (C) Effect of A20 on MYD88L265P-mediated p65 nuclear localization in 293T cells. Cells were transfected with MYD88L265P and A20 and analyzed by IF. ***P < .001. The bar represents 10 μm. MYD88, Alexa Fluor 488 (green); p65, Alexa Fluor 647 (red). (D) The effect of A20 knockdown on p65 phosphorylation and RNF138 level in RPCI.WM1 and TMD8 cells. (E) Effect of DUBs on RNF138-mediated MYD88L265P ubiquitination in 293T cells. (F) Effect of A20 and Ub mutants on MYD88L265P ubiquitination in 293T cells. (G) Effect of A20 deubiquitination activity on MYD88L265P ubiquitination in 293T cells. Cells were transfected with GFP-MYD88L265P, FLAG-RNF138, and His-Ub, along with HA-A20 or HA-A20 C103A. The ubiquitination in panels A-G was analyzed as in Figure 1E. WCE, whole-cell extract.