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