Lymphoma-associated MyD88 mutants and their isolated TIR domains constitutively hyperactivate NF-κB signaling. (A) Structure of a human MyD88 TIR monomer (Protein Data Bank accession number 2JS7) with lymphoma-associated mutations highlighted in red. The α helices are shown in beige and β sheets in light pink. The BB loop is shown in cyan. (B-F) HEK293 cells were transfected with plasmids for full-length (B-C) or TIR-domain–only (D-F) constructs of lymphoma-associated MyD88 mutants, in parallel with NF-κB–inducible firefly luciferase and constitutive Renilla luciferase reporters, and TLR4 and MD-2 plasmids (F only). Cells were harvested 24 hours later (B-E) or stimulated with LPS (100 ng/mL) for another 24 hours (F). Luciferase activity was measured by DLA (B,D,F) or separately transfected cell lysates loaded on SDS polyacrylamide gel electrophoresis (PAGE) for immunoblot (C,E). For panels B, D, and F, data presented are means + standard deviation (SD) of triplicate samples, and for panels B to F, data shown are representative of at least 3 independent experiments. EV, empty vector; IB, immunoblot.