Autophagy monitoring assay: qualitative analysis of MAP LC3-I to II conversion by immunoblot.
Lysosomal dysfunction is a recognized toxic mechanism for xenobiotics, which can result in various pathological states. There is concern that nanoparticles, in particular, may cause lysosomal pathologies, since they are likely to accumulate within lysosomes. Dysregulation of the autophagy-lysosomal degradation pathway is an example of lysosomal dysfunction associated with exposure to some nanomaterials. Here, we present a method to monitor autophagy by measurement the autophagosome marker LC3-II, a phosphatidylethanolamine (PE)-conjugated form of microtubule-associated protein 1 light chain 3-I (MAP LC3-I). As other conditions could potentially result in LC3-II expression, treatment-related changes in expression should be further evaluated by morphological assessment, using techniques such as electron microscopy, to confirm autophagosome involvement.
Nanotechnology Characterization Laboratory, Advanced Technology Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD, USA.
Christopher B
CB
McLeland
Jamie
J
Rodriguez
Stephan T
ST
Stern
D000818:Animals; D001343:Autophagy / Q000502:physiology*; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D015151:Immunoblotting / Q000379:methods*; D018374:LLC-PK1 Cells; D008027:Light; D008247:Lysosomes / Q000378:metabolism / Q000502:physiology; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000378:metabolism; D013552:Swine
D015415:Biomarkers; D008869:Microtubule-Associated Proteins; D010714:Phosphatidylethanolamines