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. 2013 Feb 7;22(10):2083–2096. doi: 10.1093/hmg/ddt058

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© The Author 2013. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — AF-6 activates parkin ubiquitin-ligase activity at the mitochondria. (A) HEK293 cells were transfected with HA-parkin and Flag-ubiquitin, in the presence of either myc-AF-6 or myc-LacZ. Lysates were fractionated into cytosolic and mitochondrial fractions, and parkin was immunoprecipitated from each fraction using an anti-HA antibody. Parkin auto-ubiquitination was determined using an anti-Flag antibody (upper panel). The levels of HA-parkin immunoprecipitates were determined using an anti-HA antibody (second panel). The levels of AF-6 and LacZ in the total cells lysates were determined using an anti-myc antibody (third and fourth panels). (B) HEK293 cells were transfected and processed as in (A). The levels of auto-ubiquitinated HA-parkin and total immunoprecipitated HA-parkin were determined by ImageMaster. The graph shows the quantification of auto-ubiquitinated HA-parkin relative to immunoprecipitated HA-parkin from cytosolic and mitochondrial fractions. Error bars represent standard error of three independent experiments. **Significantly different from control at P < 0.01.