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Analysis of relevant parameters for autophagic flux using HeLa cells expressing EGFP-LC3

AuthorsMuñoz-Braceras, Sandra ; Escalante, Ricardo
Issue Date2016
PublisherHumana Press
CitationProteostasis: 313-329 (2016)
SeriesMethods in Molecular Biology 1449
AbstractMacroautophagy (called just autophagy hereafter) is an intracellular degradation machinery essential for cell survival under stress conditions and for the maintenance of cellular homeostasis. The hallmark of autophagy is the formation of double membrane vesicles that engulf cytoplasmic material. These vesicles, called autophagosomes, mature by fusion with endosomes and lysosomes that allows the degradation of the cargo. Autophagy is a dynamic process regulated at multiple steps. Assessment of autophagy is not trivial because the number autophagosomes might not necessarily refl ect the real level of autophagic degradation, the so-called autophagic fl ux. Here, we describe an optimized protocol for the analysis of relevant parameters of autophagic fl ux using HeLa cells stably expressing EGFP-LC3. These cells are a convenient tool to determine the infl uence of the downregulation or overexpression of specifi c proteins in the autophagic fl ux as well as the analysis of autophagy-modulating compounds. Western blot analysis of relevant parameters, such as the levels of EGFP-LC3, free EGFP generated by autophagic degradation and endogenous LC3·I–II are analyzed in the presence and absence of the autophagic inhibitor chloroquine.
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