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. 2017 Jul 24;8:102. doi: 10.1038/s41467-017-00085-7

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — The binding of Nt-Arg to p62 ZZ domain facilitates disulfide bond-linked aggregation of p62 and p62–LC3 interaction. a In vitro oligomerization assay using myc/His tagged p62 deletion mutants (Supplementary Fig. 2a), followed by non-reducing SDS-PAGE and immunoblotting using a mixture of antibodies to p62 and Myc. b In vitro oligomerization assay using myc/His tagged wild type p62 and mutants carrying point mutations in ZZ domain. c In vitro p62 oligomerization assay using p62 D69A and D129A mutants in comparison with wild-type p62. d R-nsP4 pulldown assay using constructs used in c. e In vitro oligomerization assay of p62 ectopically expressed in HEK293 cells treated with 50 mM Arg-Ala in the presence of 50 mM β-mercaptoethanol (β-ME). f R-nsP4 peptide binding assay of p62 Cys mutants. Each Cys/Ala p62 mutant with myc/His tag was expressed in HEK293 cells, and 50 μg of total protein was used for pulldown. g In vitro oligomerization assay of p62 Cys mutants used in f. Cell lysates with ectopically expressed wild-type p62 and Cys mutants that can bind Nt-Arg were incubated with 20 mM RIFS tetrapeptide for 1.5 h at room temperature. h ELISA measuring the interaction of p62 ZZ domain mutants with LC3. Cell lysates overexpressing p62 proteins were incubated with Arg-Ala at different concentrations to allow p62 binding to LC3-GST linked to glutathione coated plates. Shown are the means (±S.D.) of three independent experiments, each performed in triplicate. A one-way ANOVA was performed to determine statistical significance (**P < 0.01; ****P < 0.0001). i ELISA measuring p62_D69A interaction with LC3 as described in h. The graph represents the mean (±S.D.) of three independent experiments. Statistical significance was calculated using a one-way ANOVA test (**P < 0.01; ****P < 0.0001). j Similar to i except that p62–LC3 interaction was measured in the presence of 25 mM Arg-Ala or Ala-Arg in combination with β-mercaptoethanol (β-ME) at concentrations indicated. Data represent the mean (±S.D.) of three independent experiments. Statistical significance was determined using a one-way ANOVA test (***P < 0.001; ****P < 0.0001)