YFP-WT PINK1 (green) shows mitochondrial localization in HeLa cells following treatment with the mitochondrial depolarizing agent CCCP (carbonyl cyanide m-chlorophenyl hydrazone). When fixed cells are permeabilized with 0.25% Triton X-100 (TX-100), the antibody to GFP (red) (used to identify YFP-WT PINK1) and the antibody to Tom20 (white), a mitochondrial outer membrane marker, are able to bind their epitopes. This demonstrates that when mitochondrial membrane potential is dissipated, YFP-WT PINK1 accumulates on the outer mitochondrial membrane. YFP-WT PINK1 transfected HeLa cells were treated with CCCP (10µM) for 3 hrs, fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100. Primary antibodies used were: anti-GFP polyclonal Ab (Invitrogen) and anti-Tom20 mAb (BD); secondary antibodies used were: Alexa Fluor 594 and 647. Imaging was performed on an LSM510 Meta (Carl Zeiss, Inc) with a 63x 1.4 NA oil differential interference contrast Plan Apo objective. Image contrast and brightness were adjusted in the LSM image browser (Zeiss). This image corresponds to Fig 4c, 3rd row from top, and is part of a differential permeabilization assay to determine submitochondrial localization of PINK1 and that is further described in Fig 4c of J Cell Biol, 191: 933-942, 2010. Images in Fig 4 include CIL#s 13733, 13734, 13729, 13730, 13731, 13732, 13717, 13718, 13719, 13720, 13721, 13722, 13723, 13724.
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