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. Author manuscript; available in PMC: 2013 Aug 24.
Published in final edited form as: Immunity. 2012 Aug 24;37(2):223–234. doi: 10.1016/j.immuni.2012.04.015

Fig. 4. TBK-1 co-fractionates and colocalizes with intracellular membranous organelles containing autophagic adaptors and machinery.

Fig. 4

A,B. Analysis by subcellular fractionation of Rab8b, TBK-1 and components of autophagic machinery (UVRAG, p62, LC3-II) in resting cells (Full, full medium) or upon induction of autophagy by starvation (Starve). Membranous organelles from RAW 264.7 macrophages uninduced (A) or induced (B) for autophagy were subjected to subcellular fractionation by isopycnic sucrose density gradient centrifugation, fractions collected, and proteins analyzed by immunolotting. PNS, postnuclear supernatant. 1–4, pooled fractions. Rectangle over fraction 9, convergence in autophagic organelles (LC3-II) of: Rab8b, TBK-1, UVRAG (Beclin 1 interacting protein specific for autophagosomal maturation), and autophagic adaptor p62. Refractive indexes below the lanes reflect sucrose density of each fraction. C. Images; confocal microscopy analysis of endogenous UVRAG (Alexa568) and endogenous TBK-1 (Alexa488). Cells, BMM, uninduced (Full) and induced (Starvation) for autophagy. Arrows, coloclaization; insets, enlarged areas. D. Pearson’s coefficient for TBK-1 and UVRAG colocalization (Starv, starvation). E. Analysis of TBK-1 localization relative to autophagic adaptor p62 in BMM. Images: endogenous TBK-1 (Alexa568; red), p62 (Alexa488; green) and merged. Line tracing, analysis of colocalization of TBK-1 (red tracing) and p62 (green tracing). F. Pearson’s colocalization coefficients for TBK-1-p62. (Starv, starvation). Data, means ± se (n=3, three independent experiments with at least 5 images analyzed per experiment; †, p≥0.05; **, p<0.01; ANOVA).