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. 2014 Apr 1;28(7):708–722. doi: 10.1101/gad.238246.114

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© 2014 Wu et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 6. — Hrd1 is a novel E3 ubiquitin ligase of Nrf2. (A) Schematic illustration of the amino acid sequence of the RING domain of Hrd1. Conserved cysteine residues that are mutated to serines are labeled with arrows. (B) Immunoblot analysis of cell lysates from HEK293T cells transfected with an empty vector, Flag-Hrd1, or Flag-C291S. Cells were untreated or treated with tBHQ for 16 h. (C) Cell-based ubiquitylation analysis was performed in HEK293T cells cotransfected with Nrf2, Flag-Hrd1, or Flag-C291S mutant and HA-ubiquitin for 48 h. (D) In vitro ubiquitylation of Nrf2 in the presence of Flag-Hrd1 and Flag-C291S was measured. HA-Nrf2-containing complexes were immunoprecipitated using HA beads from HEK293T cells transfected with Nrf2 and either Flag-Hrd1-WT or Flag-Hrd1-C291S. HA bead-bound proteins were incubated with purified E1, E2-UbcH5c, ubiquitin, and ATP. Following denaturation by boiling, Nrf2 was immunoprecipitated with anti-Nrf2 antibodies, and ubiquitylation of Nrf2 was detected by immunoblot analysis with an anti-ubiquitin antibody. (E) The Nrf2 protein half-life in HEK293T cells transfected with either an empty vector, Flag-Hrd1, or Flag-C291S was measured as described above. (F) Immunoblot analysis of Nrf2 in Keap1^−/− MEF cells transfected with an empty vector, Flag-Hrd1, or Flag-XBP1s for 24 h. (G) Immunoblot analysis of the indicated proteins. HEK293T cells were cotransfected with Flag-Hrd1 and either Nrf2-WT, Nrf2-ETGE/AAAA, or Nrf2-DLG/AAA. (H) Immunoblot analysis of the indicated proteins. HEK293T cells were cotransfected with Flag-Hrd1 and either Nrf2-WT, Nrf2-ETGE+2S, or Nrf2-ETGE-DLG+2S. (I) Immunoblot analysis of the indicated proteins. HEK293T cells were cotransfected with Flag-Hrd1 and either Nrf2-WT or Nrf2-K7/R7 for 24 h. (J) Immunoblot analysis of the indicated proteins. HEK293T cells were cotransfected with Flag-Hrd1 and either Nrf2-WT, Nrf2-NLS2, Nrf2-NES1, or Nrf2-NES2.