Localization of NT-UCH-L1 in mitochondria.

<p>(A) HeLa cells stably expressing UCH-L1-myc, NT-UCH-L1-myc and K15/157R NT-UCH-L1-myc were stained with anti-myc and Alexa Fluor 488 secondary antibody (green). Mitochondria and nucleus were stained with Mitotracker (red) and DAPI (blue), respectively. Cells were visualized by confocal microscopy. (B) Cells were fractionated into cytosolic and mitochondrial fractionations. UCH-L1s were immunoblotted using anti-myc antibody and Hsp60 and PRX6, a mitochondrial and cytosolic marker protein, respectively, were immunoblotted using anti-Hsp60 and anti-PRX6 antibodies. The relative distribution of UCH-L1-myc, NT-UCH-L1-myc and K15/157R NT-UCH-L1-myc in the cytosolic and mitochondrial fractions were quantified by measuring their density (C). The mean ± s.d. of three independent experiments is shown. (D) Mitochondrial fractions of HeLa cells expressing NT-UCH-L1 were incubated with various concentrations of proteinase K for 1 h at 50°C and immunoblotted with anti-myc, Tom20, cytochrome C and COX4 antibodies. (E) Cells were treated 10((M CCCP for 0, 1, or 6 h and fractionated into cytosolic and mitochondrial fractionations. UCH-L1s were immunoblotted using anti-myc antibody and Hsp60, a mitochondrial marker protein, was immunoblotted using anti-Hsp60 antibody. (F) Cells were treated with 10((M CCCP in combination with 10((M MG132 (an inhibitor of proteasomal degradation), 10 mM NH4Cl (an inhibitor of lysosomal degradation) or 10 mM 3MA (an inhibitor of autophagy). UCH-L1s were immunoblotted using anti-myc antibody.