Transcription Factor Nrf2: Examination of Nuclear Protein Levels by Immunoblotting and Promoter Response Element Binding by Chromatin Immunoprecipitation (ChIP)

Kate Petersen Shay1, Eric J. Smith2, Tory M. Hagen2

1 Linus Pauling Institute, Oregon State University, Corvallis, Oregon, 2 Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 17.13
DOI:  10.1002/0471140856.tx1713s45
Online Posting Date:  August, 2010
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Nuclear factor erythroid 2 (NF‐E2) related factor 2 (Nrf2) is a transcription factor that governs the expression of over a hundred so‐called phase II detoxification and antioxidant genes that are regulated through the antioxidant response element (ARE). Loss of Nrf2 activity has been implicated in cardiovascular disease, inflammation, aging, and cancer. Nrf2 is induced to accumulate in the nucleus when the cell encounters an oxidative stress, a fact that has been exploited experimentally to test the conditions under which ARE‐containing genes are expressed. The nuclear levels of Nrf2 give an indication of whether an experimental treatment results in Nrf2 localization and induction. mRNA levels of phase II genes may be measured as a follow‐up, but in order to show a direct link between nuclear Nrf2 accumulation and increases in gene expression, it is useful to show that Nrf2 binds to AREs in the promoters of target genes. The simplest way to do this is to employ a chromatin immunoprecipitation (ChIP) assay along with an examination of cellular Nrf2 levels by immunoblotting. Curr. Protoc. Toxicol. 45:17.13.1‐17.13.13. © 2010 by John Wiley & Sons, Inc.

Keywords: Nrf2; immunoblot; chromatin immunoprecipitation

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Table of Contents

  • Introduction
  • Basic Protocol 1: Measuring Nrf2 Levels by Immunoblotting
  • Basic Protocol 2: Nrf2 Chromatin Immunoprecipitation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
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Basic Protocol 1: Measuring Nrf2 Levels by Immunoblotting

  • Cells in culture
  • Cell culture medium
  • Nrf2‐inducing agent, e.g., buthionine sulfoximide (Sigma) or tertiary butyl hydroquinone (Sigma)
  • PBS (see recipe)
  • Hypotonic lysis buffer (see recipe)
  • 10% (v/v) Igepal or Nonidet P‐40 (NP‐40)
  • Nuclear extraction buffer (see recipe)
  • 2× SDS loading buffer (see recipe)
  • Ponceau S solution (see recipe)
  • PBS/Tween: PBS with 0.1% (v/v) Tween 20
  • Blocking buffer: PBS/Tween with 5% (w/v) nonfat dry milk
  • Primary antibodies to Nrf2 (e.g., H‐300, Santa Cruz Biotechnology)
  • Secondary antibodies, HRP‐conjugated and matched to the species of primary antibody
  • Chemiluminescence reagents
  • 6‐well tissue culture dishes
  • Cell scraper
  • 1.5‐ml microcentrifuge tubes
  • Rocking platform
  • Additional reagents and equipment for protein quantitation ( appendix 3G), SDS‐PAGE ( appendix 3F), and immunoblotting (unit 2.3)

Basic Protocol 2: Nrf2 Chromatin Immunoprecipitation

  • Dynabeads bound to protein G (Invitrogen)
  • RIPA buffer (see recipe)
  • Primary antibody to Nrf2 (e.g., H‐300, Santa Cruz Biotechnology) and control antibody
  • Frozen rat liver or cultured cells of interest
  • FTC buffer (see recipe)
  • PBS, ice cold
  • 1.25 M glycine
  • ChIP lysis buffer (see recipe)
  • 20 mg/ml proteinase K solution (see recipe)
  • ChIP RIPA buffer (see recipe)
  • TE buffer (see recipe)
  • Elution buffer (see recipe)
  • Input elution buffer (see recipe)
  • 1:1 (v/v) phenol/chloroform
  • 2.5 mg/ml linear polyacrylamide solution (see recipe)
  • Absolute and 70% ethanol, molecular biology grade, ice cold
  • Dry ice/ethanol bath
  • 0.6‐ and 1.5‐ml siliconized microcentrifuge tubes
  • Magnetic racks for 0.6‐ and 1.5‐ml microcentrifuge tubes
  • Razor blades
  • 100‐mm disposable Petri dishes
  • Sonicator
  • Thermocycler
  • Additional reagents and equipment for DNA purification ( appendix 3A) and agarose gel electrophoresis (unit 2.2)
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Literature Cited

Literature Cited
   Apopa, P.L., He, X., and Ma, Q. 2008. Phosphorylation of Nrf2 in the transcription activation domain by casein kinase 2 (CK2) is critical for the nuclear translocation and transcription activation function of Nrf2 in IMR‐32 neuroblastoma cells. J. Biochem. Mol. Toxicol. 22:63‐76.
   Dahl, J.A. and Collas, P. 2007a. Q2ChIP, a quick and quantitative chromatin immunoprecipitation assay, unravels epigenetic dynamics of developmentally regulated genes in human carcinoma cells. Stem Cells 25:1037‐1046.
   Dahl, J.A. and Collas, P. 2007b. A quick and quantitative chromatin immunoprecipitation assay for small cell samples. Front. Biosci. 12:4925‐4931.
   Dahl, J.A. and Collas, P. 2008a. MicroChIP—a rapid micro chromatin immunoprecipitation assay for small cell samples and biopsies. Nucleic Acids Res. 36:e15.
   Dahl, J.A. and Collas, P. 2008b. A rapid micro chromatin immunoprecipitation assay (microChIP). Nat. Protoc. 3:1032‐1045.
   Dhakshinamoorthy, S., Jain, A.K., Bloom, D.A., and Jaiswal, A.K. 2005. Bach1 competes with Nrf2 leading to negative regulation of the antioxidant response element (ARE)‐mediated NAD(P)H:quinone oxidoreductase 1 gene expression and induction in response to antioxidants. J. Biol. Chem. 280:16891‐16900.
   Igarashi, K. and Sun, J. 2006. The heme‐Bach1 pathway in the regulation of oxidative stress response and erythroid differentiation. Antioxid. Redox Signal. 8:107‐118.
   Itoh, K., Chiba, T., Takahashi, S., Ishii, T., Igarashi, K., Katoh, Y., Oyake, T., Hayashi, N., Satoh, K., Hatayama, I., Yamamoto, M., and Nabeshima, Y. 1997. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem. Biophys. Res. Commun. 236:313‐322.
   Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680‐685.
   Moi, P., Chan, K., Asunis, I., Cao, A., and Kan, Y.W. 1994. Isolation of NF‐E2‐related factor 2 (Nrf2), a NF‐E2‐like basic leucine zipper transcriptional activator that binds to the tandem NF‐E2/AP1 repeat of the beta‐globin locus control region. Proc. Natl. Acad. Sci. U.S.A. 91:9926‐9930.
   Nguyen, T., Nioi, P., and Pickett, C.B. 2009. The Nrf2‐antioxidant response element signaling pathway and its activation by oxidative stress. J. Biol. Chem. 284:13291‐13295.
   Shenvi, S., Dixon, B.M., Shay, K.P., and Hagen, T.M. 2008. A rat primary hepatocyte culture model for aging studies. Curr. Protoc. Toxicol. 37:14.7.1‐14.7.10.
   Sun, Z., Chin, Y.E., and Zhang, D.D. 2009. Acetylation of Nrf2 by p300/CBP augments promoter‐specific DNA binding of Nrf2 during the antioxidant response. Mol. Cell Biol. 29:2658‐2672.
   Zhu, M. and Fahl, W.E. 2001. Functional characterization of transcription regulators that interact with the electrophile response element. Biochem. Biophys. Res. Commun. 289:212‐219.
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