Measurement of autophagy in cells and tissues.
Two major proteolysis systems, the ubiquitin-proteasome system, and the autophagy-lysosome system, contribute to degradation of various types of protein and/or protein aggregates. In general, the autophagy-lysosome system is involved in bulk intracellular degradation of proteins and organelles, while the ubiquitin-proteasome system is selective. During autophagy, a cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes, and LC3-II is degraded by lysosomal hydrolases after the fusion of autophagosomes with lysosomes. Therefore, lysosomal turnover of LC3-II reflects starvation-induced autophagic activity, and detection of LC3 by immunoblotting or immunofluorescence has become a reliable method for monitoring autophagy. When autophagy is impaired, the level of p62/SQSTM1, a ubiquitin- and LC3-binding protein, is increased in addition to the accumulation of ubiquitinated proteins. Here, we describe basic protocols to analyze endogenous LC3-II, p62, and autophagy-related proteins by immunoblotting, immunofluorescence, and electron microscopy.
Tanida
Isei
I
Waguri
Satoshi
S
D000818:Animals; D001343:Autophagy; D015153:Blotting, Western; D002457:Cell Extracts; D002460:Cell Line; D002477:Cells / Q000166:cytology*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006801:Humans; D008247:Lysosomes / Q000201:enzymology / Q000648:ultrastructure; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008954:Models, Biological; D010588:Phagosomes / Q000648:ultrastructure
D002457:Cell Extracts; D008869:Microtubule-Associated Proteins