Filter trapping protocol to detect aggregated proteins in human cell lines

Chhipi-Shrestha, Jagat K.; Yoshida, Minoru; Iwasaki, Shigeo

doi:10.1016/j.xpro.2022.101571

Cited by 2 publications

(1 citation statement)

References 8 publications

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“…A serial dilution of fractionated samples was subjected to vacuum filtration through a cellulose acetate membrane (0.2 µm, Sterlitech) using a Slot Blot apparatus as described in ref. 77 . Insoluble species retained on the membrane were analyzed using anti-GFP.…”

Section: Methodsmentioning

confidence: 99%

Dynamics of DNA damage-induced nuclear inclusions are regulated by SUMOylation of Btn2

Kumar,

Mathew,

Stirling

2024

Nat Commun

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Spatial compartmentalization is a key facet of protein quality control that serves to store disassembled or non-native proteins until triage to the refolding or degradation machinery can occur in a regulated manner. Yeast cells sequester nuclear proteins at intranuclear quality control bodies (INQ) in response to various stresses, although the regulation of this process remains poorly understood. Here we reveal the SUMO modification of the small heat shock protein Btn2 under DNA damage and place Btn2 SUMOylation in a pathway promoting protein clearance from INQ structures. Along with other chaperones, and degradation machinery, Btn2-SUMO promotes INQ clearance from cells recovering from genotoxic stress. These data link small heat shock protein post-translational modification to the regulation of protein sequestration in the yeast nucleus.

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Section: Methodsmentioning

confidence: 99%

Dynamics of DNA damage-induced nuclear inclusions are regulated by SUMOylation of Btn2

Kumar,

Mathew,

Stirling

2024

Nat Commun

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Biochemical analysis to study wild-type and polyglutamine-expanded ATXN3 species

Quinet,

Paz-Cabrera,

Freire

2024

PLoS ONE

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Spinocerebellar ataxia type 3 (SCA3) is a cureless neurodegenerative disease recognized as the most prevalent form of dominantly inherited ataxia worldwide. The main hallmark of SCA3 is the expansion of a polyglutamine tract located in the C-terminal of Ataxin-3 (or ATXN3) protein, that triggers the mis-localization and toxic aggregation of ATXN3 in neuronal cells. The propensity of wild type and polyglutamine-expanded ATXN3 proteins to aggregate has been extensively studied over the last decades. In vitro studies with mass spectrometry techniques revealed a time-dependent aggregation of polyglutamine-expanded ATXN3 that occurs in several steps, leading to fibrils formation, a high status of aggregation. For in vivo experiments though, the techniques commonly used to demonstrate aggregation of polyglutamine proteins, such as filter trap assays, SDS-PAGE and SDS-AGE, are unable to unequivocally show all the stages of aggregation of wild type and polyglutamine-expanded ATXN3 proteins. Here we describe a systematic and detailed analysis of different known techniques to detect the various forms of both wild type and pathologic ATXN3 aggregates, and we discuss the power and limitation of each strategy.

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Filter trapping protocol to detect aggregated proteins in human cell lines

Cited by 2 publications

References 8 publications

Dynamics of DNA damage-induced nuclear inclusions are regulated by SUMOylation of Btn2

Dynamics of DNA damage-induced nuclear inclusions are regulated by SUMOylation of Btn2

Biochemical analysis to study wild-type and polyglutamine-expanded ATXN3 species

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