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.
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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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