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Monitoring Notch Activation in Cultured Mammalian Cells: Luciferase Complementation Imaging Assays

  • Ma. Xenia G. IlaganEmail author
  • Raphael KopanEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1187)

Abstract

Notch activation and cleavage releases the Notch intracellular domain (NICD), which translocates to the nucleus, where it associates with its DNA-binding partner CSL to recruit the coactivator MAML and additional cofactors to ultimately activate target gene expression. Taking advantage of the specific interaction between NICD and these factors, we have developed a luciferase complementation imaging (LCI)-based reporter system to quantitatively monitor Notch activation in real time in live cells. In this chapter, we describe the use of Notch LCI reporters for measuring protein interactions and performing detailed kinetic analyses of receptor activation and its responses to various stimuli.

Key words

Notch Protein complementation assay PCA Luciferase complementation imaging LCI Split luciferase Molecular imaging Bioluminescence Protein–protein interaction 

Notes

Acknowledgements

We would like to thank our colleagues in the Kopan Lab and at the Washington University Molecular Imaging Center for helpful discussions and excellent technical assistance. Special thanks go to Shuang Chen for critically reading this protocol. This work was supported by NIH grant P50 CA94056.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Developmental BiologyWashington University School of MedicineSaint LouisUSA
  2. 2.Division of Developmental Biology, Cincinnati Children’s Hospital Medical CenterUniversity of Cincinnati College of MedicineCincinnatiUSA

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