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. Author manuscript; available in PMC: 2016 Oct 1.
Published in final edited form as: Mol Cell. 2015 Sep 10;60(1):7–20. doi: 10.1016/j.molcel.2015.08.016

Figure 2. Recruitment of mitophagy adaptors OPTN NDP52, and SQSTM1, but not TAX1BP1, to depolarized mitochondria requires TBK1.<.

Figure 2

br>(A–D) The indicated HFT cells stably expressing FLAG-HA-OPTN (Panel A) or FLAG-HA-NDP52 (Panel C) were left untreated or depolarized for 1h with AO in the presence or absence of the TBK1 inhibitor MRT (2μM, pre-treatment time 1h). Cells were imaged by confocal microscopy after staining with α-HA (green), α-TOMM20 (red), and Hoechst to detect DNA (blue) (scale bar, 20 microns). Normalized MOC values for co-localization of α-HA and α-TOMM20 were determined from >50 cells as described in Experimental Procedures. Panels B and D analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. *p<0.05, **p<0.01, ****p<0.0001. n.s., not significant. Error bars represent SEM.

(E,F) TBK1 is required for efficient recruitment of OPTN, NDP52, and SQSTM1, but not TAX1BP1 to depolarized mitochondria. Assays were performed as in Panel A in HFT-PARKINWT;TBK1−/− cells (scale bar, 20 microns). Panel F displays MOC values for SQSTM1 and TAX1BP1 co-localization with TOMM20 (error bars represent SEM).

(G) Co-localization of p-TBK1S172 (green) with TOMM20-positive mitochondria (red) in depolarized HFT-PARKINWT cells was examined by immunofluorescence and confocal microscopy prior to image analysis to determine the MOC. Error bars represent SEM from triplicate experiments. (scale bar, 20 microns).

(H) FLAG-HA-OPTN localizes to a subset of mitochondrial domains occupied by p-S65 UB in response to depolarization. HFT-PARKINWT cells stably expressing FLAG-HA-OPTN were depolarized for 75 min with AO and subjected to immunofluorescence with α-HA, α-p-S65 UB, or Hoechst to detect nuclei. Scale bar, 20 microns.

(I) MOC for localization of HA-OPTN with p-S65 UB and for localization of p-S65 UB puncta with HA-OPTN puncta. Error bars are SEM of MOCs obtained from the indicated number of cell.

(J) Localization of p-S65 UB to mitochondrial domains requires PARKIN. HFT cells lacking PARKIN were depolarized for 75 min with AO and subjected to immunofluorescence with α-TOMM20, α-p-S65 UB, or Hoechst to detect nuclei. Scale bar, 20 microns. See also Figure S2.