Monitoring Notch activation in cultured mammalian cells: luciferase complementation imaging assays

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.