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dc.contributor.authorJarvela, Timothy S
dc.contributor.authorChaplot, Kriti
dc.contributor.authorLindberg, Iris
dc.date.accessioned2021-02-19T21:28:10Z
dc.date.available2021-02-19T21:28:10Z
dc.date.issued2021-01-26
dc.identifier.urihttp://hdl.handle.net/10713/14734
dc.description.abstractAlpha-synuclein pre-formed fibrils (PFFs) represent a promising model system for the study of cellular processes underlying cell-to-cell transmission of alpha-synuclein proteopathic aggregates. However, the ability to differentiate the fate of internalized PFFs from those which remain in the extracellular environment remains limited due to the propensity for PFFs to adhere to the cell surface. Removal of PFFs requires repeated washing and/or specific quenching of extracellular fluorescent PFF signals. In this paper we present a new method for analyzing the fate of internalized alpha-synuclein. We inserted a tobacco etch virus (TEV) protease cleavage site between alpha-synuclein and green fluorescent protein and subjected cells to brief treatment with TEV protease after incubation with tagged PFFs. As the TEV protease is highly specific, non-toxic, and active under physiological conditions, protection from TEV cleavage can be used to distinguish internalized PFFs from those which remain attached to the cell surface. Using this experimental paradigm, downstream intracellular events can be analyzed via live or fixed cell microscopy as well as by Western blotting. We suggest that this method will be useful for understanding the fate of PFFs after endocytosis under various experimental manipulations.en_US
dc.description.urihttps://doi.org/10.1371/journal.pone.0241161en_US
dc.language.isoenen_US
dc.publisherPublic Library of Scienceen_US
dc.relation.ispartofPLoS ONEen_US
dc.subjectPFFen_US
dc.subject.meshTEV proteaseen_US
dc.subject.meshalpha-Synucleinen_US
dc.subject.meshCell Membraneen_US
dc.subject.meshTobacco etch virusen_US
dc.subject.meshGreen Fluorescent Proteinsen_US
dc.subject.meshMicroscopyen_US
dc.subject.meshBlotting, Westernen_US
dc.titleA protease protection assay for the detection of internalized alpha-synuclein pre-formed fibrils.en_US
dc.typeArticleen_US
dc.identifier.doi10.1371/journal.pone.0241161
dc.identifier.pmid33497415
dc.source.volume16
dc.source.issue1
dc.source.beginpagee0241161
dc.source.endpage
dc.source.countryUnited States
dc.source.countryUnited States


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