Fig. 2

The active form of TBK1 localizes at the Golgi apparatus after TLR3 stimulation. a HEK293T cells stably expressing HA-TLR3 were either left untreated or stimulated with poly(I:C) (10 μg/mL) for 30 or 60 minutes. Cells 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. b, c HEK293T cells stably expressing HA-TLR3 were either left untreated (control) or stimulated with poly(I:C) (10 μg/mL) for 1 h (+ Poly(I:C)). The indicated proteins were then analyzed by immunofluorescence. The Golgi apparatus was stained with an antibody against GM130, and an antibody against EEA1 was used to stain endosomes. Scale bars, 10 μm. In b, on the right, enlargement of the framed zone in the overlay. d Crude heavy membrane fractions from unstimulated or poly(I:C)-treated HEK293T cells stably expressing HA-TLR3 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. * Indicates non-specific bands. e Increased concentrations of mitochondria- (P5) or Golgi (P25)-enriched fractions of unstimulated (Unst) or poly(I:C)-treated HEK293T cells stably expressing HA-TLR3 were incubated with recombinant GST-IRF3 in the presence of ATP. The degree of IRF3 phosphorylation was determined by immunoblotting