监测培养的哺乳动物细胞中的Notch激活：荧光素酶互补成像分析。

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

作者信息

Ilagan Ma Xenia G, Kopan Raphael

机构信息

Department of Developmental Biology, Washington University School of Medicine, Saint Louis, MO, 63110, USA,

出版信息

Methods Mol Biol. 2014;1187:155-68. doi: 10.1007/978-1-4939-1139-4_12.

DOI:10.1007/978-1-4939-1139-4_12

PMID:25053488

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.

摘要

Notch激活和切割会释放Notch细胞内结构域（NICD），NICD会转运至细胞核，在细胞核中它与DNA结合伴侣CSL结合，招募共激活因子MAML和其他辅助因子，最终激活靶基因表达。利用NICD与这些因子之间的特异性相互作用，我们开发了一种基于荧光素酶互补成像（LCI）的报告系统，以在活细胞中实时定量监测Notch激活。在本章中，我们描述了使用Notch LCI报告基因来测量蛋白质相互作用，并对受体激活及其对各种刺激的反应进行详细的动力学分析。