Fig. 1.

Effects of TBK1 ALS mutations on its kinase activity. (A) TBK1 autophosphorylation and IRF3 activation in overexpression experiments. Various expression constructs for TBK1 ALS mutations were transfected into TBK1 knockout 293T cells; 24 h later, total cell lysates were prepared, and the levels of pS172 TBK1, total TBK1, pS386 IRF3, total IRF3, and MAP kinase p42/44 were determined by western blots with specific antibodies. Positions of TBK1 ALS mutations in this study are indicated in the human TBK1 protein diagram. Note that TBK1 L11S was not detected by the total TBK1 antibody because of epitope disruption in the mutant protein. Groups I to III mutants are color coded, and familial mutations are labeled with asterisks. (B) Effects of mutations on the IFNβ-luciferase reporter assays. Expression constructs for various TBK1 mutations were cotransfected with an IFNβ promoter-driven firefly luciferase construct and a renilla luciferase reference construct into TBK1 knockout 293T cells; luciferase activities were measured 24 h after transfection. (C) Effects of TBK1 ALS mutations on NFκB reporter activity. Similar to experiments in B but a 2-copy PRDII element of the IFNβ enhancer-driven firefly luciferase construct was used instead of the IFNβ reporter.