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Transactivation and Coactivator Recruitment Assays for Measuring Farnesoid X Receptor Activity

  • Chia-Wen (Amy) Hsu
  • Jinghua Zhao
  • Menghang XiaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1473)

Abstract

The farnesoid X receptor (FXR) is a nuclear receptor responsible for homeostasis of bile acids, lipids, and glucose. Compounds that alter endogenous FXR signaling can be used as therapeutic candidates or identified as potentially hazardous compounds depending on exposure doses and health states. Therefore, there is an increasing need for high-throughput screening assays of FXR activity to profile large numbers of environmental chemicals and drugs. This chapter describes a workflow of FXR modulator identification and characterization. To identify compounds that modulate FXR transactivation at the cellular level, we first screen compounds from the Tox21 10 K compound library in an FXR-driven beta-lactamase reporter gene assay multiplexed with a cell viability assay in the same well of the 1536-well plates. The selected compounds are then tested biochemically for their ability to modulate FXR-coactivator binding interactions using a time-resolved fluorescence resonance energy transfer (TR-FRET) coactivator assay. The assay results from the workflow can be used to prioritize compounds for more extensive investigations.

Key words

Farnesoid X receptor Nuclear receptor Drugs Environmental chemicals High-throughput screening HTS Reporter gene Beta-lactamase Coactivator recruitment Fluorescence resonance energy transfer FRET 

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Chia-Wen (Amy) Hsu
    • 1
  • Jinghua Zhao
    • 1
  • Menghang Xia
    • 1
    Email author
  1. 1.National Center for Advancing Translational SciencesNational Institutes of HealthBethesdaUSA

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