Using enhanced-mitophagy to measure autophagic flux.
Bottom Line: As a result, there is much interest in understanding the dynamics of autophagy in a variety of situations.As such, these assays do not measure changes in endogenous cargo degradation which is the ultimate end-point of the autophagy process.We consider therefore that this assay will prove to be a valuable resource for investigations in which autophagy is considered important and is believed to be modulated.
Affiliation: Cancer Research UK Beatson Institute, Garscube Estate, Switchback Rd, Glasgow G61 1BD, UK.Show MeSH
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Mentions: To test our hypothesis that enhanced-mitophagy could be utilized to measure autophagic flux, we employed a commercial panel of antibodies which enables Western blotting for a number of mitochondrial proteins at the same time. 3T3-SA cells which had been infected with retrovirus expressing Parkin fused to the yellow fluorescent protein (YFP-Parkin) and parental control cells were treated with antimycin A (an inhibitor of the electron transport chain) and oligomycin (an inhibitor of ATP synthase) for 2 days. Cell lysates were then prepared and examined by Western blotting. This revealed that over this time frame there was no noticeable depletion of mitochondrial proteins in control cells whereas all mitochondrial proteins were dramatically depleted in cells expressing exogenous YFP-Parkin (Fig. 2).
Affiliation: Cancer Research UK Beatson Institute, Garscube Estate, Switchback Rd, Glasgow G61 1BD, UK.