Fig. 4.

TBK1 variants exhibit differing kinetics and affinities with damaged mitochondria. (A) Representative confocal sections of live HeLa cells depleted of endogenous TBK1 expressing Parkin (green) and TBK1 variants (magenta), treated with CCCP for up to 90 min. White box (Inset) indicates a single representative event tracked over time to measure recruitment of Parkin and TBK1. Stills from timelapse are shown in the panels. Time is indicated as min:sec from initial Parkin recruitment. (Scale bars: zoom out, 10 μm; zoom in, 2 μm.) (B) Background-subtracted TBK1 signal intensity tracked over time with respect to Parkin half maximum (0, vertical dotted line). n = 3 to 6 representative events from at least three independent experiments. Error bars indicate SEM. (C) A representative Western blot of HEK TBK1^−/− cells expressing the respective TBK1 variants, treated with CCCP or vehicle, and enriched for mitochondria (Left) or cytosol (Right). Quantification was carried out on Mito fractions to compare association of the respective TBK1 variants and Parkin with mitochondria. Numbers to the left of blots indicate kDa. (D) Quantification of C with Parkin (Top) and TBK1 (Bottom) bands normalized to TOM20 and compared to average level of WT-TBK1–expressing cells treated with CCCP (dotted line). *P ≤ 0.05, **P < 0.01 by ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Error bars indicate SEM. n = 3 independent experiments.