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. 2015 Apr 13;209(1):111–128. doi: 10.1083/jcb.201410050

Figure 3.

Figure 3.

Ubiquitin chains recruit phosphomimetic Parkin to energized mitochondria in cells. (A) HeLa cells expressing GFP chimeras of WT Parkin or a phosphomimetic (S65E) mutant were treated with MG-132 (10 µM, 3 h), stained with the ΔΨm-dependent dye TMRE, and observed with a fluorescence microscope. (B) HeLa cells coexpressing GFP chimeras of WT Parkin or a phosphomimetic (S65E) mutant with a WT or phosphomimetic (S65D) Mt-4×Ub (tandem tetra-ubiquitin fused with a targeting signal for mitochondrial outer membrane) were observed as in A. (C) Cells expressing GFP-Parkin(S65E) with WT or phosphomimetic (S65D) Mt-4×Ub were subjected to immunocytochemistry using an anti-V5 antibody that detects Mt-4×Ub. (D) HeLa cells coexpressing the indicated GFP-Parkin mutants and Mt-4×Ub were treated with MG-132 (10 µM, 3 h), stained with TMRE, and observed with a fluorescence microscope. Bars, 10 µm.