Detection of Autophagy in Caenorhabditis elegans by Western Blotting Analysis of LGG-1.

Cold Spring Harbor Protocols
Nicholas J Palmisano, Alicia Meléndez

Abstract

A common way to measure the induction of autophagy in yeast and mammalian cells is to compare the amount of Atg8/LC3-I with that of Atg8-PE/LC3-II by using western blot analysis. This is because changes in the amount of LC3-II correlate closely with changes in the number of autophagosomes present in cells. Atg8/LC3 is initially synthesized as an unprocessed form, which is proteolytically processed to form Atg8/LC3-I, and then this is modified into the phosphatidylethanolamine (PE)-conjugated Atg8-PE/LC3-II form. Atg8/LC3-II is membrane bound, whereas Atg8-PE/LC3-I is cytosolic. By associating with both the inner and outer membranes of the autophagosome, Atg8-PE/LC3-II is the only autophagy reporter that is reliably associated with completed autophagosomes. In the nematode Caenorhabditis elegans, the ortholog of Atg8/LC3 is LGG-1. Here, we discuss how changes in the levels of LGG-1-II (and the paralog LGG-2) protein can, with appropriate controls, be used to monitor autophagy activity in nematodes and present a protocol for monitoring changes in the protein levels of different forms of LGG-1 by western blotting.

References

Feb 26, 2000·Molecular Biology of the Cell·C BucciB van Deurs
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Jul 6, 2007·Autophagy·Noboru Mizushima, Tamotsu Yoshimori
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Nov 16, 2011·Autophagy·Shensi ShenGuido Kroemer
Mar 6, 2012·Journal of Cell Science·Abderazak DjeddiRenaud Legouis

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Citations

Sep 5, 2017·Cells·Yanfang ChenRenaud Legouis
Aug 9, 2019·Journal of Experimental & Clinical Cancer Research : CR·Lei YinBo Peng
Feb 3, 2016·Cold Spring Harbor Protocols·Nicholas J Palmisano, Alicia Meléndez

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