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  • Characterization of macroautophagic flux in vivo using a leupeptin-based assay.

Characterization of macroautophagic flux in vivo using a leupeptin-based assay.

Autophagy (2011-04-05)
Jeffrey Haspel, Rahamthulla S Shaik, Emeka Ifedigbo, Kiichi Nakahira, Tamas Dolinay, Joshua A Englert, Augustine M K Choi
ABSTRACT

Macroautophagy is a highly conserved catabolic process that is crucial for organ homeostasis in mammals. However, methods to directly measure macroautophagic activity (or flux) in vivo are limited. In this study we developed a quantitative macroautophagic flux assay based on measuring LC3b protein turnover in vivo after administering the protease inhibitor leupeptin. Using this assay we then characterized basal macroautophagic flux in different mouse organs. We found that the rate of LC3b accumulation after leupeptin treatment was greatest in the liver and lowest in spleen. Interestingly we found that LC3a, an ATG8/LC3b homologue and the LC3b-interacting protein p62 were degraded with similar kinetics to LC3b. However, the LC3b-related proteins GABARAP and GATE-16 were not rapidly turned over in mouse liver, implying that different LC3b homologues may contribute to macroautophagy via distinct mechanisms. Nutrient starvation augmented macroautophagic flux as measured by our assay, while refeeding the animals after a period of starvation significantly suppressed flux. We also confirmed that beclin 1 heterozygous mice had reduced basal macroautophagic flux compared to wild-type littermates. These results illustrate the usefulness of our leupeptin-based assay for studying the dynamics of macroautophagy in mice.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Acid phosphatase Assay Kit, 1 kit sufficient for 1,000 assays (multiwell plates), 1 kit sufficient for 100 assays (tubes)
Sigma-Aldrich
Anti-p62/SQSTM1 antibody produced in rabbit, ~1 mg/mL, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Cycloheximide, ≥95% (HPLC)
Sigma-Aldrich
SIGMAFAST Protease Inhibitor Cocktail Tablets, EDTA-Free, for use in purification of Histidine-tagged proteins
Sigma-Aldrich
Puromycin dihydrochloride from Streptomyces alboniger, ≥98% (HPLC), powder
Sigma-Aldrich
Chloroquine diphosphate salt, powder or crystals, 98.5-101.0% (EP)