title: Detection of Autophagy in Caenorhabditis elegans by Western Blotting Analysis of LGG-1.
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.
authors.affiliation: Department of Biology, Queens College-CUNY, Flushing, New York 11367; The Graduate Center, The City University of New York, New York 10016.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Palmisano
authors.affiliation: Department of Biology, Queens College-CUNY, Flushing, New York 11367; The Graduate Center, The City University of New York, New York 10016.
authors.forename: Alicia
authors.initials: A
authors.lastname: Meléndez
mesh_terms: D000818:Animals; D001343:Autophagy; D015153:Blotting, Western / Q000379:methods*; D017173:Caenorhabditis elegans / Q000166:cytology / Q000502:physiology*; D020868:Gene Silencing; D008967:Molecular Biology / Q000379:methods*; D061848:Optical Imaging / Q000379:methods*; D010274:Parasitology / Q000379:methods*
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