title: Renilla Luciferase-LC3 Based Reporter Assay for Measuring Autophagic Flux.
abstract: Macroautophagy (autophagy) is a dynamic intracellular degradation pathway. Monitoring the flux through the autophagy pathway is experimentally challenging but obviously a prerequisite for the proper investigation of the process. Here, we present an indirect autophagy flux assay based on monitoring the degradation of an autophagosome-associated fusion protein Rluc-LC3 by luminescence detection. The method is suitable for screening purposes with a high number of parallel samples and can be used for measurements in cell lysates as well as in living cells. The Rluc-LC3 assay has proven useful for the identification of genes, miRNAs, and small molecules that regulate autophagy flux in mammalian cells.
authors.affiliation: Cell Death and Metabolism, Center for Autophagy, Recycling and Disease, Danish Cancer Society Research Center, Copenhagen, Denmark.
authors.forename: T
authors.initials: T
authors.lastname: Farkas
authors.affiliation: Cell Death and Metabolism, Center for Autophagy, Recycling and Disease, Danish Cancer Society Research Center, Copenhagen, Denmark. Electronic address: mj@cancer.dk.
authors.forename: M
authors.initials: M
authors.lastname: Jäättelä
mesh_terms: D000818:Animals; D001343:Autophagy; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D049410:Luciferases, Renilla / Q000032:analysis* / Q000378:metabolism; D061986:MCF-7 Cells; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D011993:Recombinant Fusion Proteins; D049410:Luciferases, Renilla
keywords: Autophagy; High-throughput screening assay; LC3; Luciferase; Real time; Signaling