Assessing the functionality of both mutant TBK1 alleles in the response to transfected poly(I:C) and ssRNA (7sk-as). (a) TBK1^−/− MEFs were either left untransfected (NT) or were transfected with a mock vector, WT TBK1, or mutant constructs: D50A (mutation present in P2) or G159A (the mutation in P1). After 24 h, the cells were stimulated by transfection with Lipofectamine of 25 µg/ml poly(I:C) or 22.5 ng/µl ssRNA (7sk-as). ELISA was performed to assess IFN-β and IL-6 production 24 h after stimulation. The experiment shown is representative of three independent experiments. (b) TBK1 expression from the transfected constructs was assessed by Western blotting with an antibody against TBK1. β-Tubulin was used as a loading control. (c) IL-6 production as assessed by ELISA after stimulation with 10 ng/ml IL-1β for 24 h after transfection in TBK1^−/− MEFs either not transfected (NT) or transfected with a mock vector or a vector encoding WT TBK1 or the mutants G159A and D50A TBK1. The experiment shown is representative of three independent experiments. NS, nonstimulated.