GFP-ATG8 plasmid熒光表達質粒載體

examining autophagosome completion using GFP-Atg8 as a marker for protease protection.
GFP-Atg8 shows the same behavior as Atg8, and autophagic flux can be followed by monitoring the vacuolar delivery and subsequent breakdown of GFP-Atg8. When autophagic flux is normal, GFP-Atg8 that is present inside the autophagosome is cleaved after the autophagic body membrane is lysed and the contents are exposed to vacuolar hydrolases. The proteolysis of GFP-Atg8 releases an intact GFP moiety, which accumulates in the vacuole as autophagy proceeds, because it is relatively resistant to degradation. The increase in free GFP levels can be detected and quantified by western blot analysis and correlated with the autophagic rate.5-8 Alternatively, the degradation of the nonselective cargo Pgk1-GFP,9 or the quantitative Pho8Δ60 assay can be used to monitor the magnitude of autophagy.


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