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. 2010 Nov 29;191(5):933–942. doi: 10.1083/jcb.201008084

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Figure 2. — The 52-kD form of endogenous PINK1 is found inside mitochondria and does not recruit Parkin. (a) HeLa cells were initially treated with 50 µM MG132 for 10 h and then together with 10 µM CCCP for a final 3 h. Cells were fractionated and analyzed by immunoblotting using antibodies against the indicated proteins. (b) The mitochondrial fraction from panel a was subjected to alkaline extraction using sodium carbonate and immunoblotted for PINK1, Cyt c, and Tim23. (c) Mitochondria from panel a were incubated for 30 min on ice with various concentrations of PK followed by immunoblotting using antibodies against PINK1 and the indicated mitochondrial markers. (d) HeLa cells stably expressing YFP-Parkin were treated with DMSO, 10 µM CCCP, or 10 µM MG132 for 3 h followed by staining with TMRE and confocal imaging. Bars, 20 µm. Blue arrowheads, FL PINK1; red arrowheads, 52-kD PINK1.