Electrophoretic Mobility Shift Assay (EMSA) and Supershift Assay of Cytochrome P450 2B6 in Response to Estrogen

Part of the Methods in Molecular Biology book series (MIMB, volume 1366)

Abstract

Electrophoretic mobility shift assay (EMSA) is an invaluable tool to study interaction of proteins with DNA. Estrogens are major female hormones and modulate biological function through estrogen receptor (ER). ER regulates its target gene expression via the classical mechanism in which ER directly binds to its target gene promoter or the nonclassical mechanism involving tethering of ER to other transcription factors (such as AP-1 proteins). Here, we describe the EMSA to examine the nonclassical mechanism of ER action in regulation of a gene CYP2B6 by using competition and supershift assays.

Key words

EMSA Supershift Estrogen Estrogenreceptor AP-1 CYP2B6 

References

  1. 1.
    Fried M, Crothers DM (1981) Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res 9(23):6505–6525CrossRefGoogle Scholar
  2. 2.
    Koh KH, Jurkovic S, Yang K et al (2012) Estradiol induces cytochrome P450 2B6 expression at high concentrations: implication in estrogen-mediated gene regulation in pregnancy. Biochem Pharmacol 84(1):93–103CrossRefGoogle Scholar
  3. 3.
    Pedram A, Razandi M, O'Mahony F, Harvey H, Harvey BJ, Levin ER (2013) Estrogen reduces lipid content in the liver exclusively from membrane receptor signaling. Sci Signal 6(276):ra36CrossRefGoogle Scholar
  4. 4.
    Bjornstrom L, Sjoberg M (2005) Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes. Mol Endocrinol 19(4):833–842CrossRefGoogle Scholar
  5. 5.
    Safe S, Kim K (2008) Non-classical genomic estrogen receptor (ER)/specificity protein and ER/activating protein-1 signaling pathways. J Mol Endocrinol 41(5):263–275CrossRefGoogle Scholar
  6. 6.
    Larouche K, Bergeron MJ, Leclerc S, Guerin SL (1996) Optimization of competitor poly(dI-dC).poly(dI-dC) levels is advised in DNA-protein interaction studies involving enriched nuclear proteins. Biotechniques 20(3):439–444PubMedGoogle Scholar
  7. 7.
    Lambertini E, Tavanti E, Torreggiani E, Penolazzi L, Gambari R, Piva R (2008) ERalpha and AP-1 interact in vivo with a specific sequence of the F promoter of the human ERalpha gene in osteoblasts. J Cell Physiol 216(1):101–110CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Internal MedicineRush University Medical CenterChicagoUSA
  2. 2.Departments of Pharmacy Practice and Biopharmaceutical Sciences, College of PharmacyUniversity of Illinois at ChicagoChicagoUSA

Personalised recommendations