γ-Secretase Epsilon-cleavage Assay
Ting-Hai Xu1,2,3, Yan Yan1,2,3, Kaleeckal G Harikumar4
1Key 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.
Abstract:
γ-Secretase epsilon-cleavage assay is derived from the cell-based Tango assay (Kang et al., 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.
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