LC3 and Autophagy.
Microtubule-associated protein 1A/1B-light chain 3 (LC3) is a soluble protein with a molecular mass of approximately 17 kDa that is distributed ubiquitously in mammalian tissues and cultured cells. During autophagy, autophagosomes engulf cytoplasmic components, including cytosolic proteins and organelles. Concomitantly, a cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes. Autophagosomes fuse with lysosomes to form autolysosomes, and intra-autophagosomal components are degraded by lysosomal hydrolases. At the same time, LC3-II in autolysosomal lumen is degraded. Thus, lysosomal turnover of the autophagosomal marker LC3-II reflects starvation-induced autophagic activity, and detecting LC3 by immunoblotting or immunofluorescence has become a reliable method for monitoring autophagy and autophagy-related processes, including autophagic cell death. Here we describe basic protocols to assay for endogenous LC3-II by immunoblotting, immunoprecipitation, and immunofluorescence.
Tanida
Isei
I
Department of Biochemistry, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan.
Ueno
Takashi
T
Kominami
Eiki
E
D000818:Animals; D001343:Autophagy / Q000502:physiology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D047468:Immunoprecipitation; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D010588:Phagosomes / Q000378:metabolism
C480860:Map1lc3b protein, mouse; D008869:Microtubule-Associated Proteins