Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

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

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 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/.

PMC Copyright notice

Figure 5. — Hrd1 and Nrf2 interact directly. (A) Immunoprecipitation analysis was performed using cell lysates from HEK293T cells cotransfected with Flag-Hrd1 and hemagglutinin (HA)-tagged Nrf2 (HA-Nrf2). (B) Immunoprecipitation analysis was performed using cell lysates from HEK293T cells, immunoprecipitated by normal rabbit IgG or anti-Nrf2 antibody. (C) Schematic illustration of glutathione S-transferase (GST)-tagged Nrf2-WT and its deletion mutants used for interaction domain mapping. (D) Sepharose affinity chromatography analysis of the interaction between Nrf2 and Hrd1. [³⁵S]-Hrd1-WT was generated by in vitro transcription and translation. GST-tagged Nrf2-WT and its indicated deletion mutants were expressed and purified from Escherichia coli cells. Equal amounts of Nrf2 proteins were used for each pull-down assay. (E) Immunoprecipitation analysis was performed using cell lysates of HEK293T cells cotransfected with Flag-Hrd1 and either HA-Nrf2, HA-Nrf2-ΔNeh4, HA-Nrf2-ΔNeh5, or HA-Nrf2-ΔNeh4-5. (F) Immunoblot analysis was performed with cell lysates from HEK293T cells cotransfected with Flag-Hrd1 and either Nrf2-WT or Nrf2-ΔNeh4-5 for 24 h. (G) Immunoprecipitation of Nrf2 by Flag-Hrd1 and its mutants. HEK293T cells were transfected with Flag-Hrd1 and the indicated truncation mutants. Hrd1 proteins were first immunoprecipitated using Flag-M2 beads and then incubated with His-tagged Nrf2 that was expressed and purified from E. coli cells. Equal amounts of Hrd1 proteins were used for each experiment.