Figure 1. Parkin accumulates on impaired... | Rockefeller University Press

Figure 1.

$Figure 1. Parkin accumulates on impaired mitochondria. (a and b) HEK293 cells treated with DMSO control (a) or 10 μM CCCP (b) for 1 h immunostained for endogenous Parkin (green) and a mitochondrial marker, Tom20 (red). The bottom panels show enlarged views of the boxed areas. Arrows indicate mitochondria that colocalize with endogenous Parkin. (c and d) HEK293 cells (c) and rat cortical neurons (d) depolarized with CCCP for 1 and 5 h, respectively. Cells were immunoblotted for endogenous Parkin. PNS, HM, and PHM indicate postnuclear supernatant, mitochondrial-rich heavy membrane pellet, and post–heavy membrane supernatant, respectively. VDAC is a mitochondrial marker. (e) HeLa cells expressing YFP-Parkin (green) treated with DMSO, 10 μM CCCP, or 10 μM CCCP + 10 μM oligomycin for 1 h. Cells were stained for the mitochondrial marker cytochrome c (red). Line scans below the images indicate colocalization between Parkin (green) and mitochondria (red) and correlate to the lines drawn in the images. (f) YFP-Parkin colocalization with mitochondria scored for ≥300 cells per condition in at least two experiments. (g) YFP-Parkin accumulation in mitochondrial fraction assessed as in panel c. Numbers to the right of the gel blots indicate molecular weight standards in kD. (h) HeLa cells treated with 2 mM paraquat or paraquat + 10 mM N-acetyl-cysteine (NAC) for 24 h scored for colocalization, as in panel f. Error bars indicate standard deviation of at least three replicates. Bars, 10 μm.$

Parkin accumulates on impaired mitochondria. (a and b) HEK293 cells treated with DMSO control (a) or 10 μM CCCP (b) for 1 h immunostained for endogenous Parkin (green) and a mitochondrial marker, Tom20 (red). The bottom panels show enlarged views of the boxed areas. Arrows indicate mitochondria that colocalize with endogenous Parkin. (c and d) HEK293 cells (c) and rat cortical neurons (d) depolarized with CCCP for 1 and 5 h, respectively. Cells were immunoblotted for endogenous Parkin. PNS, HM, and PHM indicate postnuclear supernatant, mitochondrial-rich heavy membrane pellet, and post–heavy membrane supernatant, respectively. VDAC is a mitochondrial marker. (e) HeLa cells expressing YFP-Parkin (green) treated with DMSO, 10 μM CCCP, or 10 μM CCCP + 10 μM oligomycin for 1 h. Cells were stained for the mitochondrial marker cytochrome c (red). Line scans below the images indicate colocalization between Parkin (green) and mitochondria (red) and correlate to the lines drawn in the images. (f) YFP-Parkin colocalization with mitochondria scored for ≥300 cells per condition in at least two experiments. (g) YFP-Parkin accumulation in mitochondrial fraction assessed as in panel c. Numbers to the right of the gel blots indicate molecular weight standards in kD. (h) HeLa cells treated with 2 mM paraquat or paraquat + 10 mM N-acetyl-cysteine (NAC) for 24 h scored for colocalization, as in panel f. Error bars indicate standard deviation of at least three replicates. Bars, 10 μm.