Both mutant TBK1 alleles are loss-of-function but through different mechanisms. (a) In vitro kinase assays: substrate (Akt) phosphorylation by both mutant kinases, G159A and D50A, compared with the phosphorylation levels for WT TBK1 and the kinase-dead (KD) K38M TBK1. A single experiment representative of three independent experiments performed is shown. HEK293T cells were transfected with WT and mutant constructs or left untransfected (NT). Black lines indicate that intervening lanes have been spliced out. IP, immunoprecipitation. (b) TBK1^−/− MEFs were either left untransfected (NT) or were transfected with a mock vector, WT TBK1, or mutant constructs: D50A (mutation present in P2), G159A (the mutation in P1), and S172A (kinase-dead, KD). After 24 h, the cells were stimulated with 10 or 50 µg/ml poly(I:C). ELISA was performed to assess IFN-β and IL-6 production 24 h after stimulation. The data shown are representative of three independent experiments. (c) TBK1 expression from the transfected constructs was assessed by Western blotting with antibodies against TBK1, with β-tubulin used as a loading control. (d) TBK1^−/− MEFs were either not transfected (NT) or transfected with a mock vector or a vector encoding WT TBK1 or the mutants, G159A, D50A, or S172A. 24 h later, cells were stimulated with 10 ng/ml IL-1β. IL-6 production was measured by ELISA. The data shown are representative of three independent experiments. NS, nonstimulated.