K63-linked polyubiquitination of MYD88^L265P is critical for NF-κB activation. (A) MYD88^L265P and MYD88^L265P-5KR ubiquitination by RNF138 in 293T cells. Ubiquitination was analyzed as in Figure 1E. (B) Co-IP analysis of the interaction between RNF138 and MYD88^L265P or MYD88^L265P-5KR in 293T cells. (C) Co-IP analysis of the interaction between FLAG-MYD88^WT and GFP-tagged MYD88^WT, MYD88^L265P, or MYD88^L265P-5KR in 293T cells. Equimolar amounts of MYD88 constructs were introduced into the cells. (D) Co-IP analysis of the interaction between IRAK-1 and MYD88^L265P or MYD88^L265P-5KR in 293T cells. (E) IB analysis of p65 phosphorylation in RPCI.WM1 cells with MYD88 knockdown and reintroduction of MYD88^L265P or MYD88^L265P-5KR. WCE, whole-cell extract.