A20 inhibits MYD88^L265P 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 MYD88^L265P /MYD88^WT, RNF138, and DUBs and examined for luciferase activity. Blue spot, A20; gray spot, vector control. (C) Effect of A20 on MYD88^L265P-mediated p65 nuclear localization in 293T cells. Cells were transfected with MYD88^L265P 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 MYD88^L265P ubiquitination in 293T cells. (F) Effect of A20 and Ub mutants on MYD88^L265P ubiquitination in 293T cells. (G) Effect of A20 deubiquitination activity on MYD88^L265P ubiquitination in 293T cells. Cells were transfected with GFP-MYD88^L265P, 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.