Functional assessment of lysosomal Rab7 and RILP with RNA interference and overexpression in Spodoptera frugiperda Sf9 cell lines

The interaction manner and biological function of Rab7 and its effector, Rab-interacting lysosomal protein (RILP), remain unclear in invertebrates. We provide a protocol for detecting the effects of Rab7 and RILP terminals on lysosome and autophagy in Spodoptera frugiperda Sf9 cells with overexpress...

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Detalles Bibliográficos
Autores principales: Cui, Gaofeng, Jiang, Zhiyan, Zhong, Guohua
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10598039/
https://www.ncbi.nlm.nih.gov/pubmed/37851568
http://dx.doi.org/10.1016/j.xpro.2023.102646
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author Cui, Gaofeng
Jiang, Zhiyan
Zhong, Guohua
author_facet Cui, Gaofeng
Jiang, Zhiyan
Zhong, Guohua
author_sort Cui, Gaofeng
collection PubMed
description The interaction manner and biological function of Rab7 and its effector, Rab-interacting lysosomal protein (RILP), remain unclear in invertebrates. We provide a protocol for detecting the effects of Rab7 and RILP terminals on lysosome and autophagy in Spodoptera frugiperda Sf9 cells with overexpression and RNA interference. We describe steps for overexpressing plasmids, generating long double-stranded RNA, and transfecting them into Sf9 cells. We then detail procedures for cell immunofluorescence imaging with harmine treatment and fluorescence analysis. For complete details on the use and execution of this protocol, please refer to Cui et al. (2023).(1)
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spelling pubmed-105980392023-10-26 Functional assessment of lysosomal Rab7 and RILP with RNA interference and overexpression in Spodoptera frugiperda Sf9 cell lines Cui, Gaofeng Jiang, Zhiyan Zhong, Guohua STAR Protoc Protocol The interaction manner and biological function of Rab7 and its effector, Rab-interacting lysosomal protein (RILP), remain unclear in invertebrates. We provide a protocol for detecting the effects of Rab7 and RILP terminals on lysosome and autophagy in Spodoptera frugiperda Sf9 cells with overexpression and RNA interference. We describe steps for overexpressing plasmids, generating long double-stranded RNA, and transfecting them into Sf9 cells. We then detail procedures for cell immunofluorescence imaging with harmine treatment and fluorescence analysis. For complete details on the use and execution of this protocol, please refer to Cui et al. (2023).(1) Elsevier 2023-10-17 /pmc/articles/PMC10598039/ /pubmed/37851568 http://dx.doi.org/10.1016/j.xpro.2023.102646 Text en © 2023 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Cui, Gaofeng
Jiang, Zhiyan
Zhong, Guohua
Functional assessment of lysosomal Rab7 and RILP with RNA interference and overexpression in Spodoptera frugiperda Sf9 cell lines
title Functional assessment of lysosomal Rab7 and RILP with RNA interference and overexpression in Spodoptera frugiperda Sf9 cell lines
title_full Functional assessment of lysosomal Rab7 and RILP with RNA interference and overexpression in Spodoptera frugiperda Sf9 cell lines
title_fullStr Functional assessment of lysosomal Rab7 and RILP with RNA interference and overexpression in Spodoptera frugiperda Sf9 cell lines
title_full_unstemmed Functional assessment of lysosomal Rab7 and RILP with RNA interference and overexpression in Spodoptera frugiperda Sf9 cell lines
title_short Functional assessment of lysosomal Rab7 and RILP with RNA interference and overexpression in Spodoptera frugiperda Sf9 cell lines
title_sort functional assessment of lysosomal rab7 and rilp with rna interference and overexpression in spodoptera frugiperda sf9 cell lines
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10598039/
https://www.ncbi.nlm.nih.gov/pubmed/37851568
http://dx.doi.org/10.1016/j.xpro.2023.102646
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