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. 2017 Aug 19;88(10):869–875. doi: 10.1136/jnnp-2017-316174

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In vitro functional characterisation of TBK1 missense variants. (A) Subcellular distribution of TBK1 mutants visualised by anti-FLAG staining in differentiated motor neuron-like NSC-34 cells expressing the indicated tagged constructs. Similarly to wild-type TBK1, mutants localise in the cytosol, vesicular structures and lamellar domains at the cell periphery. L59Ffs*16-TBK1 is undetected. (B) Expression levels and kinase activity of TBK1 mutants (Top panels). Western blot analysis of FLAG-tagged wild-type TBK1 and indicated variants expressed in human embryonic kidney (AD293) cells along with HA-tagged IRF3. Green fluorescent protein (GFP) transfected cells serve as control (mock). Quantification of steady-state protein levels of TBK1 mutants relative to wild-type and normalised to GADPH is reported below each lane. The steady-state protein levels are lower for the p.Arg357Gln and slightly higher for the p.Ile397Thr mutants. The truncating mutant p.Leu59Phefs*16 abolishes TBK1 expression. Phosphorylation of IRF3 on Ser-396 in the same protein lysates was revealed with a phospho-specific antibody (bottom panels). The ratio between p-IRF3 and total IRF3 is reported under the lanes (p-IRF3/tot IRF3) and these values are further normalised to the expression levels of each TBK1 mutant protein (p-IRF3/TBK1). Phosphorylation of IRF3 is impaired by the p.Asp118Asn mutation and enhanced by the p.Ile397Thr. (C) TBK1 mutants interact with OPTN. FLAG-tagged constructs of wild-type TBK1 and indicated missense variants (or GFP control plasmid; ‘mock’) were expressed in AD293 cells together with HA-tagged OPTN. TBK1 constructs were pull-down with anti-FLAG antibody; OPTN in the immunocomplexes was revealed with anti-HA antibody. Membranes were stripped and reprobed with anti-FLAG antibody to detect the immunoprecipitated TBK1 constructs. Arrows mark target proteins, whereas asterisks point to non-specific bands. (In the figure aminoacids are indicated with the one-letter code instead of the three-letter code). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HA, haemagglutinin; OPTN, optineurin; p-IRF3, phosphorylated IRF3; TBK1, TANK-binding kinase 1; WT, wild type; IB: Immunoblot.