Mutational analysis of the Mal-TIR domain.

<p>(<b>A</b>) Selective mutants of solvent-exposed residues that are highly conserved across different species and used in the NF-κB reporter assay. (<b>B</b>) Alanine scanning of each residue of the AB loop (except alanine and glycine) for the NF-κB reporter assay. Black bars indicate decreased (less than 50%) NF-κB activity. RLU: relative luciferase unit; Luc: firefly luciferase activity; Ren: Renilla luciferase activity. GST pull-down assay for the MyD88-TIR and Mal-TIR variants (<b>C</b>) and for the TLR4-TIR and Mal-TIR variants (<b>D</b>). MyD88-TIR and TLR4-TIR were purified as GST fusion proteins. Mal-TIR was double-tagged with His₆ and Myc and purified with Ni-NTA. The resulting complexes were analyzed by SDS-PAGE and Western blotting. (<b>E</b>) Bar graph of Mal-TIR wild type and mutants (E108A and F117A) binding to MyD88-TIR (left) or TLR4-TIR (right). The error bars indicate the standard error of the mean (n = 3 separate experiments). * indicates a P value<0.05, ** indicates a P value<0.001.