Figure 5: TBK1 directly binds to and phosphorylates CEP170 and NuMA.

(a) Detection of co-localization of TBK1 and CEP170 as well as TBK1 and NuMA using proximity ligation assay (PLA). Green fluorescent foci represent co-localization of TBK1 and CEP170 or TBK1 and NuMA. TBK1 antibody alone or TBK1 antibody along with IgG were used as negative controls. (b) Immunoprecipitation–western blot analysis showing the interaction of endogenous TBK1 with endogenous CEP170 and NuMA in lysates of asynchronously growing H1650 cells. (c) Immunoprecipitation–western blot analysis of H460 cell lysates showing the binding of TBK1 with NuMA and CEP170 when released from G1/S block. This interaction was inhibited when cells were released from double-thymidine block in the presence of BX795. (d,e) In vitro binding of full-length TBK1, the kinase domain of TBK1 (1–294) or the TBK1 C terminus (TBK1-284-729) with CEP170 (d) or NuMA (e). The data presented are representative of three independent experiments. Lysate/ input lane represents 20% of the input ³⁵S-methionine-labelled TBK1 used in the pull-down assay. (f) Immunoprecipitation–western blot analysis showing inhibition in serine and threonine phosphorylation of CEP170 and NuMA when TBK1 was depleted following transduction of cells with lentivirus expressing shRNA targeting TBK1. (g) In vitro kinase assays showing the phosphorylation of NuMA and CEP170 by TBK1. Coomassie brilliant blue (CBB) staining shows the amount of protein in each reaction.