PINK1-YFP R98F (green) is localized to mitochondria in the absence of the mitochondrial depolarizing agent CCCP (carbonyl cyanide-m-chlorophenyl hydrazone). The R98F mutation in PINK1-YFP partially inhibits proteolytic cleavage. When fixed cells are permeabilized with 0.25% Triton X-100 (TX-100), the antibody to GFP (red) (used to identify PINK1-YFP R98F) and the antibody to Tom20 (white), a mitochondrial outer membrane marker, are both able to bind their epitopes. This demonstrates that PINK1 R98F is located inside mitochondria. PINK1-YFP R98F transfected HeLa cells were treated with DMSO 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, bottom row, and is part of a differential permeabilization assay to determine submitochondrial localization of PINK1 R98F 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.