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. 2010 Oct 2;285(49):38214–38223. doi: 10.1074/jbc.M110.101469

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© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 6. — Failure to mono-ubiquitinating Bcl-2 and regulating autophagy by disease-linked RING1 and RING2 domain mutants. A and B, disease-linked RING1 and RING2 domain mutants (K161N, T240R, C431F, and P437L) fail to repress autophagy. HeLa cells expressing FLAG, FLAG-parkin, or disease-linked parkin mutants were subjected to immunoblot analysis with antibodies as indicated. The band densities of LC3-II, Bcl-2, p62, or Beclin 1 relative to that of GAPDH were shown. The values are the means ± S.E. from three independent experiments. **, p < 0.01; *, p < 0.05; Δ, p > 0.05, one-way ANOVA. C, disease-linked parkin mutants fail to mono-ubiquitinate Bcl-2. 293 cells expressing a combination of EGFP-Bcl-2 and HA-ubiquitin together with FLAG-parkin or its RING mutants (K161N, T240R, C431F, and P437L) were subjected to immunoprecipitation using anti-GFP antibody. Immunoprecipitants and inputs were detected with antibodies as indicated. IP, immunoprecipitation.