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. 2012 Aug 27;209(9):1567–1582. doi: 10.1084/jem.20111316

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© 2012 Herman et al.

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Figure 3. — The G159A TBK1 allele of P1 abolishes TLR3-mediated IFN induction in the patient’s fibroblasts. (a) IRF-3 phosphorylation in fibroblasts from P1, as assessed by Western blotting after stimulation with 25 µg/ml poly(I:C) for 0, 1, or 2 h, with β-tubulin as a loading control. Comparison with control cells (C+) and UNC-93B^−/− and NEMO^−/− fibroblasts. IRF3 dimerization was assessed by native Western blotting. Blots are representative of three independent experiments. Two different healthy control cell lines were tested and gave the same result. (b) Activated p65 levels, as determined by EMSA-ELISA after 30 min of stimulation with IL-1β or 1 h of poly(I:C) in control cells (C+; averaged from two control cell lines), in fibroblasts from P1 and from UNC-93B^−/− and NEMO^−/− patients. (c) IFN-β, IFN-λ, and IL-6 production, as assessed by ELISA, after 24 h of stimulation with poly(I:C) or poly(A:U) (polyadenylic-polyuridylic acid) or transfection of poly(I:C) mediated by Lipofectamine (Lipo) in control (C+) cells (averaged from two distinct control cell lines) and in fibroblasts from P1 and from UNC-93B^−/− and NEMO^−/− patients. (d) Induction of mRNA for IFNB1, IL29, and IL6 after stimulation with 25 µg/ml poly(I:C), as assessed by RT-qPCR in fibroblasts from P1, an UNC-93B^−/− patient, and controls (C+; averaged from two distinct control cell lines). Graphs present the mean values ± SD of three independent experiments for IFNB1 and IL29. The graph presented for IL6 is representative of two independent experiments. (e) Fibroblasts from P1 were untransfected (NT), mock-transfected, or transfected with a vector encoding FLAG-tagged WT TBK1 and stimulated for 24 h with the indicated reagents (poly(I:C) or poly(I:C) transfection mediated by Lipofectamine). IFN-β and IFN-λ production was assessed by ELISA. (b, c, and e) Values represent mean values ± SD calculated from three independent experiments. (f) Western blot of transfected P1 cells with anti-TBK1 and anti-FLAG antibodies. NS, nonstimulated.