Journal: The Journal of Experimental Medicine
Article Title: A TBK1 variant causes autophagolysosomal and motoneuron pathology without neuroinflammation in mice
doi: 10.1084/jem.20221190
Figure Lengend Snippet: Characterization of the TBK1 E696K variant. (A) Scheme of LuTHy-BRET assay to investigate binding of wt TBK1 and TBK1 E696K to optineurin (OPTN) or the TRAF family member–associated NF-κB activator (TANK) in live HEK293 cells. (B and C) Binding of wt TBK1- and TBK1 E696K -mCitrine-Protein A (-mCit-PA) to NanoLuc (NL)-tagged optineurin (B) or TANK (C) in LuTHy-BRET donor saturation assays. (D) Quantification of cBRET signals from TBK1 binding assays. PA-mCit-NL tandem construct shown as positive and cotransfection of single NL- and PA-mCit-tags as negative BRET controls. Relative BRET ratios were obtained by normalizing the BRET signal of optineurin or TANK with each TBK1 mutant to the interaction signals with wt TBK1, respectively. Mean ± SEM of n = 4 technical replicates per condition from three independent experiments; one-way ANOVA with Sidak’s post hoc test; **P < 0.01. (E) Comparison of wt TBK1- and TBK1 E696K -mCit-PA fusion protein expression as measured from fluorescence intensities. Mean ± SEM of n = 7 technical replicates per condition from three independent experiments; Student’s t test. (F) Western blot showing expression of the TBK1 mutants in HEK293 cells after transfection. Source data are available for this figure: .
Article Snippet: LuTHy control vectors expressing only NL (#113442; Addgene) or PA-mCit ( #113443; Addgene) were used for the calculation of corrected scores, the PA-mCit-NL tandem construct (#113444; Addgene) as positive, and NL cotransfected with PA-mCit-only as negative controls.
Techniques: Variant Assay, Bioluminescence Resonance Energy Transfer, Binding Assay, Construct, Cotransfection, Mutagenesis, Comparison, Expressing, Fluorescence, Western Blot, Transfection
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