Parkin-dependent accumulation of phosphorylated ubiquitin on depolarized mitochondria. (A) Preparation of the anti-phosphorylated ubiquitin antibody, α-pUb. Non-phosphorylated ubiquitin control (lanes 1 and 3) and ubiquitin phosphorylated by recombinant TcPINK1 (lanes 2 and 4) were subjected to Phos-tag PAGE. The membrane was separated in two at the broken line and immunoblotted with an anti-ubiquitin antibody (P4D1) or the anti–phospho-ubiquitin antibody, α-pUb. α-pUb specifically reacts with phosphorylated ubiquitin. Note that mobility does not reflect the molecular weight of proteins in Phos-tag PAGE (Kinoshita et al., 2012) and thus molecular weight markers are not shown. (B) Specificity of α-pUb in cytochemical experiments. HeLa cells expressing Mt-4×Ub ± CCCP were stained with α-pUb and Hsp60 (mitochondrial marker). Mt-4×Ub containing aggregated mitochondria were detected by α-pUb only after CCCP treatment. (C) HeLa cells expressing sole GFP (1), GFP-Parkin (2), or PINK1-GFP (3) were treated with CCCP and were stained with α-pUb. Phosphorylated ubiquitin is detectable after overproduction of Parkin or PINK1; however, restricted accumulation of phosphorylated ubiquitin on mitochondria is dependent on Parkin. (D) The number of cells with no signal (blue), a mitochondria-localized signal (red), or a cytosolic signal (green) for phosphorylated ubiquitin was determined in 100 cells. Bars represent the mean ± SD (error bars) values of three experiments. Bars, 10 µm.