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Cellular Ca2+-Responding Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-palindromic NFAT-Response Elements

  • Wei Zhang
  • Terunao TakaharaEmail author
  • Takuya Achiha
  • Hideki Shibata
  • Masatoshi MakiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1929)

Abstract

Luciferase reporter gene systems based on the NFAT-response element (RE) have been used to monitor intracellular Ca2+ elevation. However, Ca2+ mobilization agent (e.g., ionomycin) alone is not adequate to activate the currently often employed reporter gene that contains the NFAT-RE found in the IL2 promoter. In addition to activation of NFAT through the Ca2+-calmodulin/calcineurin pathway, activation of AP-1 as a partner transcription factor is essential for the IL2-based NFAT-RE system. Here, we describe a detailed method for the recently developed new reporter gene system containing the NFAT-RE from the IL8 promoter. This system enables us to monitor endpoint effects of Ca2+-mobilizing agonists independent of AP-1 activation.

Key words

Calcineurin Calcium signaling Calmodulin Carbachol Luminescence NanoLuc NFAT Reporter gene assay Transcription factor 

Notes

Acknowledgments

This work was supported by JSPS KAKENHI Grant Number 17H03803 (Grant-in-Aid for Scientific Research (B) to M.M.). We thank current and past members of the Laboratory of Molecular and Cellular Regulation for valuable suggestions and discussion.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Applied Molecular Biosciences, Graduate School of Bioagricultural SciencesNagoya UniversityNagoyaJapan

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