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. 2016 May 31;113(24):E3349–E3358. doi: 10.1073/pnas.1523810113

Fig. S5.

Fig. S5.

(A and B) Inhibition of TBK1 by 1-h pretreatment with 1 μM BX795 or expression of a phosphodeficient TBK1-S172A mutant does not block TBK1 recruitment to depolarized mitochondria at 90 min post-CCCP. (C) Inhibition of TBK1 blocks GFP-LC3 recruitment at 90 min post-CCCP, but does not block upstream recruitment of mCherry-Parkin or Halo-OPTN. (D, Top) Treatment for 90 min with 10 μM antimycin along with 10 μM oligomycin induces recruitment of LC3 and OPTN to fragmented mitochondria. (D, Bottom) LC3 recruitment, but not OPTN recruitment, is blocked by pretreatment with 1 μM BX795. (E) Immunoblot of TBK1 and actin-loading control. Treatment of HeLa cells with 20 nM TBK1 siRNA for 48 h results in a >95% reduction in TBK1 protein levels. (F) At 90 min post-CCCP, the percentage of mitochondria positive for LC3 in Parkin/OPTN-expressing cells is not affected by DMSO (Vehicle) treatment or expression of a scrambled siRNA. [Scale bars: D (full size), 10 μm; A–D (zoom), 5 μm.] Bars represent mean ± SEM.