Measurement of autophagy in cells and tissues

Methods Mol Biol. 2010:648:193-214. doi: 10.1007/978-1-60761-756-3_13.

Abstract

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.

MeSH terms

  • Animals
  • Autophagy*
  • Blotting, Western
  • Cell Extracts
  • Cell Line
  • Cells / cytology*
  • Electrophoresis, Polyacrylamide Gel
  • Fluorescent Antibody Technique / methods*
  • Humans
  • Lysosomes / enzymology
  • Lysosomes / ultrastructure
  • Mice
  • Microtubule-Associated Proteins / metabolism
  • Models, Biological
  • Phagosomes / ultrastructure

Substances

  • Cell Extracts
  • Microtubule-Associated Proteins