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Measurement of Autophagy in Cells and Tissues

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Protein Misfolding and Cellular Stress in Disease and Aging

Part of the book series: Methods in Molecular Biology ((MIMB,volume 648))

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.

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Abbreviations

Atg:

Autophagy-related product

BSA:

Bovine serum albumin

DMSO:

Dimethylsulfoxide

IMPase:

Myoinositol monophosphatase

PE:

Phosphatidylethanolamine

PI3K:

Phosphoinositide 3-kinase

LC3/MAP1LC3B:

Microtubule-associated protein 1 light chain 3, a yeast Atg8 homolog

3-MA:

3-Methyladenine

TBS:

Tris-buffered saline

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Author information

Authors and Affiliations

  1. Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo, Japan

    Isei Tanida

  2. Department of Anatomy and Histology, Fukushima Medical University School of Medicine, Fukushima, Japan

    Satoshi Waguri

Authors
  1. Isei Tanida
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  2. Satoshi Waguri
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Corresponding author

Correspondence to Isei Tanida .

Editor information

Editors and Affiliations

  1. Aarhus University Hospital, Research Unit for Molecular Medicine, University of Aarhus, Brendstrupgaardsvej 100, Aarhus N, 8200, Denmark

    Peter Bross

  2. Aarhus University Hospital, Research Unit for Molecular Medicine, University of Aarhus, Brendstrupgaardsvej 100, Aarhus N, 8200, Denmark

    Niels Gregersen

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Tanida, I., Waguri, S. (2010). Measurement of Autophagy in Cells and Tissues. In: Bross, P., Gregersen, N. (eds) Protein Misfolding and Cellular Stress in Disease and Aging. Methods in Molecular Biology, vol 648. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-756-3_13

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  • DOI: https://doi.org/10.1007/978-1-60761-756-3_13

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-755-6

  • Online ISBN: 978-1-60761-756-3

  • eBook Packages: Springer Protocols

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