γ-分泌酶ε切割分析
γ-Secretase Epsilon-cleavage Assay.
作者信息
Xu Ting-Hai, Yan Yan, Harikumar Kaleeckal G, Miller Laurence J, Melcher Karsten, Xu H Eric
机构信息
Key Laboratory of Receptor Research, VARI-SIMM Center, Center for Structure and Function of Drug Targets, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China.
出版信息
Bio Protoc. 2017 Nov 20;7(22). doi: 10.21769/BioProtoc.2900.
γ-Secretase epsilon-cleavage assay is derived from the cell-based Tango assay (Kang ., 2015), and is a fast and sensitive method to determine the initial cleavage of C99 by γ-secretase. In this protocol, we use HTL cells, which are HEK293 cells with a stably integrated luciferase reporter under the control of the bacterial tetO operator element, in which C99 C terminally fused to a reversed tetracyclin-inducible activator (rTA) transcriptional activator is expressed. Endogenous or transfected γ-secretase cleaves a C terminally fused rTA transcriptional activator from C99, allowing rTA to move to the nucleus to activate a luciferase reporter gene as a measurement for γ-secretase cleavage activity.
γ-分泌酶ε切割测定法源自基于细胞的Tango测定法（Kang等人，2015年），是一种快速且灵敏的方法，用于确定γ-分泌酶对C99的初始切割。在本实验方案中，我们使用HTL细胞，它是在细菌tetO操纵元件控制下稳定整合了荧光素酶报告基因的HEK293细胞，其中C99在C末端融合了反向四环素诱导激活剂（rTA）转录激活因子并表达。内源性或转染的γ-分泌酶从C99上切割C末端融合的rTA转录激活因子，使rTA转移到细胞核中以激活荧光素酶报告基因，作为γ-分泌酶切割活性的一种测量方法。
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