RNF138 interacts with MYD88^L265P and promotes MYD88^L265P ubiquitination. (A) Co-IP analysis of MYD88^L265P and RNF138 interaction in 293T cells transfected with the indicated constructs. (B) Direct binding between RNF138 and MYD88^L265P. FLAG-MYD88^L265P immunoprecipitated from 293T cells bound to GST-RNF138 in vitro. #, non-specific signal. (C) Co-IP analysis of the endogenous MYD88 and RNF138 interaction in lymphoma cells. (D) PLA of MYD88 and RNF138 interaction in lymphoma cells. ***P < .001. The bar represents 5 μm. Wheat germ agglutinin (WGA), Alexa Fluor 488 (green); PLA, Alexa Fluor 568 (red). (E) In vivo MYD88^L265P and MYD88^WT ubiquitination by RNF138 in 293T cells. His-Ub–linked proteins were purified using Ni-NTA beads from cell lysates before they were subjected to IB analysis with MYD88 antibody. (F) MYD88^L265P ubiquitination by RNF138 in vitro. FLAG-MYD88^L265P was incubated with E1, E2, and ubiquitin+GST or GST-RNF138 in a cell-free system. CBB Coomassie brilliant blue. (G) RNF138-mediated MYD88^L265P ubiquitination with ubiquitin mutants in 293T cells. Cells were transfected with RNF138 and MYD88^L265P, along with WT or mutant ubiquitin and subjected to Ni-NTA bead purification and IB analysis as in panel E. (H) Endogenous MYD88 ubiquitination in MYD88^L265P (RPCI.WM1, HBL-1, and TMD8 cells) and MYD88^WT (U2932 and BJAB) cell lines. Lysates from cells were immunoprecipitated using the antibody against MYD88 before they were subjected to IB with Ub-K63 antibody. (I) Endogenous MYD88^L265P ubiquitination in RPCI.WM1, TMD8, and HBL-1 cells with RNF138 knockdown. Lysates from control and RNF138 stable knockdown cells were immunoprecipitated using the antibody against MYD88 before they were subjected to IB. (J) RNF138-mediated ubiquitination on MYD88 mutants in 293T cells. His-Ub–linked proteins were purified using Ni-NTA beads from cell lysates before IB analysis with GFP antibody. WCE, whole-cell extract.