Merck

Quantifying autophagy using novel LC3B and p62 TR-FRET assays.

PloS one (2018-03-20)
Alberto Bresciani, Maria Carolina Spiezia, Roberto Boggio, Cristina Cariulo, Anja Nordheim, Roberta Altobelli, Kirsten Kuhlbrodt, Celia Dominguez, Ignacio Munoz-Sanjuan, John Wityak, Valentina Fodale, Deanna M Marchionini, Andreas Weiss
ABSTRACT

Autophagy is a cellular mechanism that can generate energy for cells or clear misfolded or aggregated proteins, and upregulating this process has been proposed as a therapeutic approach for neurodegenerative diseases. Here we describe a novel set of LC3B-II and p62 time-resolved fluorescence resonance energy transfer (TR-FRET) assays that can detect changes in autophagy in the absence of exogenous labels. Lipidated LC3 is a marker of autophagosomes, while p62 is a substrate of autophagy. These assays can be employed in high-throughput screens to identify novel autophagy upregulators, and can measure autophagy changes in cultured cells or tissues after genetic or pharmacological interventions. We also demonstrate that different cells exhibit varying autophagic responses to pharmacological interventions. Overall, it is clear that a battery of readouts is required to make conclusions about changes in autophagy.

MATERIALS
Product Number
Brand
Product Description

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Anti-Mouse IgG (whole molecule)–Peroxidase antibody produced in goat, affinity isolated antibody, buffered aqueous solution
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Anti-p62/SQSTM1 antibody produced in rabbit, ~1 mg/mL, affinity isolated antibody, buffered aqueous solution
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Anti-LC3 antibody produced in rabbit, ~1 mg/mL, affinity isolated antibody, buffered aqueous solution