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Measuring Nonselective and Selective Autophagy in the Liver.

Authors
  • Ueno, Takashi1
  • Komatsu, Masaaki2
  • 1 Laboratory of Proteomics and Biomolecular Science, Research Support Center, Juntendo University Graduate School of Medicine, Tokyo, Japan. [email protected] , (Japan)
  • 2 Department of Physiology, Juntendo University Graduate School of Medicine, Tokyo, Japan. [email protected] , (Japan)
Type
Published Article
Journal
Methods in molecular biology (Clifton, N.J.)
Publication Date
Jan 01, 2019
Volume
1880
Pages
535–540
Identifiers
DOI: 10.1007/978-1-4939-8873-0_34
PMID: 30610720
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Administration of leupeptin, a specific inhibitor of lysosomal cysteine proteinases, to starved rats or mice inhibits autolysosomal protein degradation and results in accumulation of autolysosomes in their livers. Immunoblotting of liver homogenates to examine autophagic flux in vivo reveals elevated levels of the selective autophagy substrate p62 and the autophagosomal membrane protein LC3-II in the livers of leupeptin-treated animals. Percoll density gradient centrifugation can be used to isolate autolysosomes from the livers of untreated and leupeptin-treated animals. Moreover, autolysosomes can be examined for the presence of sequestered cytoplasmic proteins as well as degradation intermediates.

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