A20 mediates RNF138 degradation through K48-linked ubiquitination. (A) Effect of A20 on RNF138 protein levels in 293T cells. (B) The half-life of RNF138 in the presence of A20. FLAG-RNF138 and GFP-A20 were cotransfected into 293T cells and treated with cycloheximide (CHX). FLAG-RNF138 and GFP-A20 protein levels were analyzed by IB, quantified by densitometry, and plotted against treatment time. (C) Effect of A20 on RNF138 polyubiquitination in 293T cells. (D) A20-mediated RNF138 polyubiquitination in the presence of MG132. 293T cells were transfected with various constructs and treated with MG132 (20 μM) for 6 hours. (E) The types of polyubiquitination chains on RNF138 in 293T cells. (F) The half-life of RNF138 with A20 knockdown in RPCI.WM1 cells. Cells were transfected with shCtrl or shA20 and treated with CHX. Endogenous RNF138 protein levels were analyzed by IB, quantified by densitometry, and plotted against treatment time. (G) Endogenous RNF138 and MYD88^L265P ubiquitination in RPCI.WM1 and TMD8 cells after A20 knockdown. (H) Effect of A20 and RNF138 K158R on MYD88^L265P ubiquitination in 293T cells. In panels C, D, E, and H, His-Ub-linked proteins were purified by using Ni-NTA beads from cell lysates before they were subjected to IB with RNF138 antibody. (I) Effect of RNF138 K158R and A20 on NF-κB reporter activity mediated by MYD88^L265P in 293T-NF-κB-Luc cells. ***P < .001. WCE, whole-cell extract.