(A) HEK293T cells were transiently transfected with IFN-β promoter firefly, β-actin promoter renilla reporter plasmids, and 100 ng of vector and plasmids encoding the full-length (WT) and truncated P1 F105fs*19 IKKε to measure IFN-β gene expression indicated by luciferase activity after 16 hours. (B) p-IRF3 (S396) and total IRF3 in whole-cell lysates from HEK293T cells expressing empty vector, WT, and P1 variant IKKε. IKKε _{(N/C-term epi)}, antibodies targeting epitopes in the N- and C-terminal parts of IKKε. (C) Lysates from HEK293T cells transfected with the indicated expression plasmids were immunoprecipitated with anti-FLAG and subjected to assays determining IKKε kinase activity. Data are presented as normalized levels of luciferase signals from the ATP measurements. (D and E) HEK293T cells were transfected with IFN-β promoter firefly, β-actin promoter renilla reporter plasmids, WT IKBKE, WT TBK1, and the indicated amounts of P1 IKBKE. (F) HEK293T cells were transfected with P1 IKBKE, and the indicated amounts of plasmids encoding FLAG-TBK1 and FLAG-IKKε. Cells were lysed 24 hours later and immunoblotted for p-TBK1 (S172), p-IKKε (S172), FLAG, IKKε (probing of only low molecular range of blotted membrane), and Vinculin. (G) HEK293T cells were transfected with 6x-His-WT or P1 IKBKE, FLAG-TBK1, and FLAG-IKBKE. Lysates were immunoprecipitated with anti-His and immunoblotted with anti-FLAG. (H–J) Control fibroblasts were transduced with lentiviral vectors encoding patient IKKε variant F105fs*19 (MOI: 12), infected with HSV-2 at the indicated MOI for 6 hours (H) or 24 hours (J), or transfected with dsDNA (I) for 6 hours. Data presented are from 1 representative of 6 (A, D, and E), 2 (F and G), and 3 (B, C, and H–J) independently performed experiments. Nonparametric Mann-Whitney rank-sum test was used for statistical analysis. Error bars represent SEM. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. NU = (Luc_max – Luc_measured)/Luc_max (see Methods).