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Using enhanced-mitophagy to measure autophagic flux.

Baudot AD, Haller M, Mrschtik M, Tait SW, Ryan KM - Methods (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Beatson Institute, Garscube Estate, Switchback Rd, Glasgow G61 1BD, UK.

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Expression of exogenous Parkin enables mitophagy. 3T3-SA cells with or without expression of YFP-Parkin were treated with 1 μM antimycin A + 1 μM oligomycin and analyzed by Western Blotting after 48 h using the MitoProfile Membrane integrity WB Antibody Cocktail. Actin was used as a loading control. The immunoblots shown are representative of what was seen in at least three independent experiments.
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f0010: Expression of exogenous Parkin enables mitophagy. 3T3-SA cells with or without expression of YFP-Parkin were treated with 1 μM antimycin A + 1 μM oligomycin and analyzed by Western Blotting after 48 h using the MitoProfile Membrane integrity WB Antibody Cocktail. Actin was used as a loading control. The immunoblots shown are representative of what was seen in at least three independent experiments.

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).


Using enhanced-mitophagy to measure autophagic flux.

Baudot AD, Haller M, Mrschtik M, Tait SW, Ryan KM - Methods (2014)

Expression of exogenous Parkin enables mitophagy. 3T3-SA cells with or without expression of YFP-Parkin were treated with 1 μM antimycin A + 1 μM oligomycin and analyzed by Western Blotting after 48 h using the MitoProfile Membrane integrity WB Antibody Cocktail. Actin was used as a loading control. The immunoblots shown are representative of what was seen in at least three independent experiments.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4358839&req=5

f0010: Expression of exogenous Parkin enables mitophagy. 3T3-SA cells with or without expression of YFP-Parkin were treated with 1 μM antimycin A + 1 μM oligomycin and analyzed by Western Blotting after 48 h using the MitoProfile Membrane integrity WB Antibody Cocktail. Actin was used as a loading control. The immunoblots shown are representative of what was seen in at least three independent experiments.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Beatson Institute, Garscube Estate, Switchback Rd, Glasgow G61 1BD, UK.

Show MeSH