プロトコール

Protocols for autophagy analysis

Mammal

・Overview: autophagy methods in mammals

・Induction of autophagy in mammalian cells

・Long-lived protein degradation

・LC3 flux assay

・p62/SQSTM1-based biochemical methods

・Proteinase K protection assay

・LC3 immunostaining

・Live imaging analysis of GFP-Atg proteins

・Electron microscopy analysis of autophagosome-related structures

・Measurement of autophagy flux using fluorescent-tagged LC3
　　- mRFP-GFP tandem fluorescent-tagged LC3
　　- FACS analysis of GFP-LC3 degradation

・Mitophagy analysis (Monitoring of mitochondrial protein degradation)

・Mitophagy analysis (Measurement of mitochondrial DNA)

・Xenophagy analysis

・Lysophagy analysis

・In vivo analysis of autophagy using GFP-LC3 transgenic mouse

・LC3 and p62 immunohistochemistry

・Autophagy flux assay in mouse

Yeast

・Overview for autophagy analysis in yeast

・Cultivation of yeast cells and induction of autophagy

・Alkaline phosphatase (ALP) assay

・Fluorescent microscopy analysis of the pre-autophagosomal structure (PAS)

・Fluorescent microscopy analysis of GFP-Atg8 translocation into the vacuole and observation of autophagic bodies

・GFP-Atg8 cleavage assay

・Pgk1-GFP cleavage assay

・Selective autophagy analysis in yeast（mitophagy, pexophagy, ER-phagy/nucleophagy, the Cvt pathway）

In vitro system

・Atg8/LC3 lipidation reaction