PINK1 recruits cytoplasmic Parkin to damaged mitochondria. (A) PINK1 knockout (PINK1^−/−) or control (PINK1^+/+) MEFs were transfected with HA-Parkin, treated with CCCP, and subjected to immunocytochemistry with the indicated antibodies. Higher magnification views of the boxed areas are shown in the insets. (B) The number of MEFs with Parkin localized to the mitochondria was counted as in Fig. 3 A. (C and D) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1^−/− MEFs. MEFs stably expressing GFP-Parkin were treated with CCCP for 4 h and subjected to immunoblotting (C) or for 24 h, followed by immunocytochemistry (D). IB, immunoblot; Ub, ubiquitylation. (E) The number of MEFs without anti-Tom20 antibody–detectable mitochondria was counted as in Fig. 3 A. In the example figure (left), blue arrowheads indicate cells without anti-Tom20 antibody–detectable mitochondria, and red arrowheads indicate cells harboring anti-Tom20 antibody–detectable mitochondria. (B and E) Error bars represent the mean ± SD values of least three experiments. Bars: (A) 10 µm; (D) 50 µm; (E) 150 µm.