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. 2007 Sep 10;178(6):1025–1038. doi: 10.1083/jcb.200611128

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Copyright © 2007, The Rockefeller University Press

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Figure 2. — Parkin mediates K63-linked polyubiquitination of L166P mutant DJ-1 in cooperation with the UbcH13/Uev1a E2 enzyme. (A) Lysates from transfected SH-SY5Y cells were subjected to immunoprecipitation with anti-Myc antibody followed by Western blotting. (B and C) Purified L166P mutant DJ-1 was subjected to in vitro ubiquitination in the presence of E1, E2 (UbcH13/Uev1a), GST-tagged parkin, and wild-type or mutant ubiquitin as indicated. Ubiquitinated mutant DJ-1 was detected by immunoblotting with anti-DJ-1 antibody. All experiments were replicated three times with similar results.