Simultaneous Examination of Cellular Pathways using Multiplex Hextuple Luciferase Assaying.

Current Protocols in Molecular Biology
Alejandro Sarrion-PerdigonesKoen J T Venken

Abstract

Multiplex experimentation that can assay multiple cellular signaling pathways in the same cells requires orthogonal genetically encoded reporters that report over large dynamic ranges. Luciferases are cost-effective, versatile candidates whose output signals can be sensitively detected in a multiplex fashion. Commonly used dual luciferase reporter assays detect one luciferase that is coupled to a single cellular pathway and a second that is coupled to a control pathway for normalization purposes. We have expanded this approach to multiplex hextuple luciferase assays that can report on five cellular signaling pathways and one control, each of which is encoded by a unique luciferase. Light emission by the six luciferases can be distinguished by the use of two distinct substrates, each specific for three luciferases, followed by spectral decomposition of the light emitted by each of the three luciferase enzymes with bandpass filters. Here, we present detailed protocols on how to perform multiplex hextuple luciferase assaying to monitor pathway fluxes through transcriptional response elements for five specific signaling pathways (i.e., c-Myc, NF-κβ, TGF-β, p53, and MAPK/JNK) using the constitutive CMV promoter as normalization cont...Continue Reading

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Dec 15, 2019·Nature Communications·Alejandro Sarrion-PerdigonesKoen J T Venken
Mar 10, 2020·Current Protocols in Molecular Biology·Marta Vazquez-VilarDiego Orzaez

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