Fig 1: Interaction of TBK1 with the scaffold protein TANK is crucial for IKK? protein degradation. (A) Immunoblot (IB) analysis of WT cells, TBK1-deficient cells (clone #2), and TBK1-deficient cells transduced with either Flag-tagged TBK1 WT or TBK1 L704 mutant described to be unable to interact with the scaffold proteins TANK, NAP1, and SINTBAD. One representative blot of n = 4 independent lysate preparations is shown. (B, C) Quantification of IBs from (A). TBK1 (B) and IKK? (C) protein expression levels normalized to the expression of the housekeeping protein ß-Actin are shown as the fold change of WT cells (mean ± SD of n = 4 lysate preparations). Individual values are visualized as dots. (C) One-way ANOVA followed by Dunnett’s multiple comparisons test was used to compare IKK? expression of TBK1-transduced with that of untransduced TBK1-deficient cells (ns – not significant, ** p = 0.01). (D) Type I IFN reporter gene expression in THP1 monocytes after stimulation for 24 h with the RIG-I/MDA5 ligand polyI:C (100 ng/ml), the cGAS ligands pDNA (100 ng/ml) or G3-YSD (500 ng/ml), IFNa (1000 U/ml), or vehicle only (Lipo). Shown is the mean ± SD of n = 5 independent experiments. Individual values are visualized as dots. (E) Whole-cell lysates of TBK1-deficient cells ectopically expressing Flag-tagged TBK1 WT, TBK1 L693A, TBK1 L694E, or TBK1 L704A were prepared and the TBK1 variants were immunoprecipitated (IP) with anti-Flag beads. Interacting proteins were detected by IB with the indicated antibodies. As input control, 2% of the lysate employed for immunoprecipitation was used. One representative blot of n = 3 independent experiments is shown. (F). Immunoblot (IB) analysis of WT cells, TBK1-deficient cells (clone #2), and TBK1-deficient cells transduced with either Flag-tagged TBK1 WT, TBK1 L693A mutant that specifically lacks the ability to interact with TANK, or TBK1 K694E mutant unable to interact with the scaffold proteins TANK and SINTBAD. One representative blot of n = 4 independent lysate preparations is shown. (G, H) Quantification of IBs from (F) TBK1 (G) and IKK? (H) protein expression levels normalized to the expression of the housekeeping protein ß-Actin are shown as the fold change of WT cells (mean ± SD of n = 4 lysate preparations). Individual values are visualized as dots. (H) One-way ANOVA followed by Dunnett’s multiple comparisons test was used to compare IKK? expression of TBK1-transduced with that of untransduced TBK1-deficient cells (ns – not significant, * p = 0.05).