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Fig. 4 | BMC Biology

Fig. 4

From: The Golgi apparatus acts as a platform for TBK1 activation after viral RNA sensing

Fig. 4

Ubiquitination promotes TBK1 targeting to the Golgi apparatus for activation. a HEK293T cells were transfected with an empty vector (Ev) or with plasmids encoding myc-tagged WT TBK1 (WT), TBK1K38M (K38M), or TBK1K30R/K401R (K30R/K401R). After 16 h, TBK1 activation and exogenous TBK1 expression were assessed by immunoblotting with anti-p-TBK1S172 and anti-myc antibodies, respectively. GAPDH was used as a loading control. b HEK293T cells were transfected with either an IFNβ promoter reporter or an NF-κB reporter, together with the Renilla luciferase gene as an internal control. In parallel, the cells were also transfected with an Ev or with plasmids encoding myc-tagged WT TBK1 (WT), TBK1K38M (K38M), or TBK1K30R/K401R (K30R/K401R). Luciferase assays were performed 24 h after transfection and the results were normalized against Renilla luciferase activity. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with WT TBK1 in Student’s t test). RLU, relative luminescence units. c Immunoblotting analysis of TBK1–/– MEFs reconstituted with WT TBK1, TBK1K38M (K38M), or TBK1K30R/K401R (K30R/K401R). As controls, TBK1+/+ and TBK1–/– MEFs are shown. d TBK1–/– MEFs reconstituted with WT TBK1 or mutants were either left untreated (MOCK) or transfected with HMW poly(I:C) (5 μg/mL) for 4 h (trPoly(I:C)). TBK1 aggregation was then assessed by immunofluorescence staining and counting of the aggregates. The data shown are means ± SD from three independent experiments (300 cells were counted per condition). **0.001 < P < 0.01 versus MEFs reconstituted with WT TBK1 (Student’s t test). e The reconstituted MEFs described in (c) and the initial TBK1–/– MEFs were transfected with HMW poly(I:C) (5 μg/mL) for 0, 2, and 4 h (trPoly(I:C)). IFNB1 mRNA levels were then assessed by RT-qPCR with normalization against GAPDH. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with WT TBK1-reconstituted MEFs in Student’s t test). AU, arbitrary unit

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