title: LC3- and p62-based biochemical methods for the analysis of autophagy progression in mammalian cells.
abstract: Autophagy is an intracellular degradation system that delivers cytoplasmic materials to the lysosome or vacuole. This system plays a crucial role in various physiological and pathological processes in living organisms ranging from yeast to mammals. Thus, an accurate and reliable measure of autophagic activity is necessary. However, autophagy involves dynamic and complicated processes that make it difficult to analyze. The term "autophagic flux" is used to denote overall autophagic degradation (i.e., delivery of autophagic cargo to the lysosome) rather than autophagosome formation. Immunoblot analysis of LC3 and p62/SQSTM1, among other proteins, has been widely used to monitor autophagic flux. Here, we describe basic protocols to measure the levels of endogenous LC3 and p62 by immunoblotting in cultured mammalian cells.
authors.affiliation: Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo 113-0033, Japan; Department of Physiology and Cell Biology, Tokyo Medical and Dental University, Tokyo 113-8519, Japan.
authors.forename: Peidu
authors.initials: P
authors.lastname: Jiang
authors.affiliation: Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo 113-0033, Japan. Electronic address: nmizu@m.u-tokyo.ac.jp.
authors.forename: Noboru
authors.initials: N
authors.lastname: Mizushima
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001343:Autophagy / Q000235:genetics*; D002460:Cell Line; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism; D008322:Mammals; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D000071456:Sequestosome-1 Protein
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D006360:Heat-Shock Proteins; C480860:Map1lc3b protein, mouse; D008869:Microtubule-Associated Proteins; D000071456:Sequestosome-1 Protein; C101343:Sqstm1 protein, mouse
keywords: Autophagic flux; Autophagy; Immunoblotting; LC3; SQSTM1; p62