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Fig. 1 | BMC Biology

Fig. 1

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

Fig. 1

Localization of the active form of TBK1 at the Golgi apparatus after RLR stimulation. a MEFs were either left unstimulated or infected with Sendai virus (SeV) for 6 or 8 h. MEFs were then fractionated as described in Additional file 1A, and samples were analyzed by immunoblotting with antibodies against the indicated proteins. EEA1, kinectin, LAMP2, GAPDH, syntaxin-6, and VDAC served as loading and purity controls for endosomes, the endoplasmic reticulum, lysosomes, the cytosol, the Golgi apparatus, and mitochondria, respectively. (Ub)n, polyubiquitin. * Indicates non-specific bands. be MEFs were either left unstimulated (control) or infected with SeV for 6 h (+ SeV). The indicated proteins were then analyzed by immunofluorescence. The Golgi apparatus was stained with an antibody raised against GM130, whereas the mitochondria were identified by labeling with an antibody against cytochrome c. Scale bars, 10 μm. On the right, enlargement of the framed zone in the overlay. f WT or TBK1–/– MEFs were either left unstimulated (control) or infected with SeV for 6 h (+ SeV). p-TBK1S172 staining was then analyzed by immunofluorescence. The Golgi apparatus was stained with an antibody raised against GM130. Scale bars, 10 μm. g Crude heavy membrane fractions from uninfected or SeV-infected MEFs were fractionated on OptiPrep density gradients (fractions range from 1 at the top to 4 at the bottom) and analyzed by immunoblotting with antibodies against the indicated proteins. (Ub)n, polyubiquitin. h Increased concentrations of mitochondria (P5) or Golgi (P25)-enriched fractions of unstimulated (Unst) or SeV-infected HEK293T cells were incubated with recombinant GST-IRF3 in the presence of ATP. The degree of IRF3 phosphorylation was determined by immunoblotting

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