Measuring Nonselective and Selective Autophagy in the Liver
Administration of leupeptin, a specific inhibitor of lysosomal cysteine proteinases, to starved rats or mice inhibits autolysosomal protein degradation and results in accumulation of autolysosomes in their livers. Immunoblotting of liver homogenates to examine autophagic flux in vivo reveals elevated levels of the selective autophagy substrate p62 and the autophagosomal membrane protein LC3-II in the livers of leupeptin-treated animals. Percoll density gradient centrifugation can be used to isolate autolysosomes from the livers of untreated and leupeptin-treated animals. Moreover, autolysosomes can be examined for the presence of sequestered cytoplasmic proteins as well as degradation intermediates.
Key wordsLiver Leupeptin Autolysosome Percoll Selective autophagy Nonselective autophagy
M.K. is supported by Grant-in-Aid for Scientific Research on Innovative Areas (25111006 and 25111001, to M.K) and Japan Society for the Promotion of Science (an A3 foresight program). T.U. is supported by High Technology Research Center Grant, Strategic Research Foundation at Private Universities. T.U. and M.K. are supported by Grant-in-Aid for Scientific Research on Priority Areas (18076005) and the Takeda Science Foundation.