title: Turnover of Lipidated LC3 and Autophagic Cargoes in Mammalian Cells.
abstract: Macroautophagy (usually referred to as autophagy) is the most important degradation system in mammalian cells. It is responsible for the elimination of protein aggregates, organelles, and other cellular content. During autophagy, these materials (i.e., cargo) must be engulfed by a double-membrane structure called an autophagosome, which delivers the cargo to the lysosome to complete its degradation. Autophagy is a very dynamic pathway called autophagic flux. The process involves all the steps that are implicated in cargo degradation from autophagosome formation. There are several techniques to monitor autophagic flux. Among them, the method most used experimentally to assess autophagy is the detection of LC3 protein processing and p62 degradation by Western blotting. In this chapter, we provide a detailed and straightforward protocol for this purpose in cultured mammalian cells, including a brief set of notes concerning problems associated with the Western-blotting detection of LC3 and p62.
authors.affiliation: Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED), Universidad de Extremadura, Cáceres, Spain; Facultad de Enfermería y Terapia Ocupacional, Universidad de Extremadura, Cáceres, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Rodríguez-Arribas
authors.affiliation: Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED), Universidad de Extremadura, Cáceres, Spain; Facultad de Enfermería y Terapia Ocupacional, Universidad de Extremadura, Cáceres, Spain.
authors.forename: S M S
authors.initials: SM
authors.lastname: Yakhine-Diop
authors.affiliation: Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED), Universidad de Extremadura, Cáceres, Spain; Facultad de Enfermería y Terapia Ocupacional, Universidad de Extremadura, Cáceres, Spain.
authors.forename: R A
authors.initials: RA
authors.lastname: González-Polo
authors.affiliation: Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED), Universidad de Extremadura, Cáceres, Spain; Facultad de Enfermería y Terapia Ocupacional, Universidad de Extremadura, Cáceres, Spain. Electronic address: mireianiso@hotmail.com.
authors.forename: M
authors.initials: M
authors.lastname: Niso-Santano
authors.affiliation: Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED), Universidad de Extremadura, Cáceres, Spain; Facultad de Enfermería y Terapia Ocupacional, Universidad de Extremadura, Cáceres, Spain. Electronic address: jfuentes@unex.es.
authors.forename: J M
authors.initials: JM
authors.lastname: Fuentes
mesh_terms: D000818:Animals; D001343:Autophagy / Q000502:physiology*; D015153:Blotting, Western; D002478:Cells, Cultured; D004586:Electrophoresis / Q000379:methods; D005347:Fibroblasts / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000378:metabolism*; D008967:Molecular Biology / Q000379:methods*; D000071456:Sequestosome-1 Protein / Q000378:metabolism
chemical_list: C480860:Map1lc3b protein, mouse; D008869:Microtubule-Associated Proteins; D000071456:Sequestosome-1 Protein; C101343:Sqstm1 protein, mouse
keywords: ATG5; Autophagic flux; Bafilomycin A1; LC3; Western-blotting; p62