Augmented heterodimerization of lymphoma-associated mutants and WT MyD88 TIR domain. (A-D) MyD88-deficient HEK293-I3A cells were transfected with plasmids for full-length (A-B) or TIR-domain–only (C-D) constructs of lymphoma-associated MyD88 mutants, in parallel with NF-κB–inducible firefly luciferase and a constitutive Renilla luciferase reporters. Cells were harvested 48 hours later; 1 lysate aliquot was used for luciferase activity measurement by DLA (A,C) and another for immunoblot upon 15% SDS-PAGE separation (B,D). Data presented are means + SD of triplicate samples, and data shown are representative of at least 3 independent experiments. (E-F) The L265P mutation leads to increased TIR-TIR oligomerization. LUMIER luciferase analysis from HEK293T cells transfected with Protein A–tagged and Renilla luciferase–tagged WT or L265P mutant full-length (E) or TIR-domain–only (amino acid sequence 155-294) (F) MyD88 constructs. Forty-eight hours posttransfection, cells were lysed and raw luciferase measured in 10% of the lysate sample. The remainder was used for Protein A purification on immunoglobulin G magnetic beads and subsequent measurement of bound luciferase. Data represent ratios of bound vs raw luciferase for each transfection upon subtraction of background (Protein A–only control bait) combined from 7 identical experiments. Means ± SDs are shown and differences tested using a Mann-Whitney U t test. EV, empty vector; IB, immunoblot.