An optimized protocol to detect protein ubiquitination and activation by ubiquitination assay in vivo and CCK-8 assay

E3 ubiquitin ligases play a role in protein degradation, cellular localization, and activation, and their dysregulation is associated with human diseases. Here, we present a protocol to detect IGF2BP1 ubiquitination and activation by an E3 ubiquitin ligase FBXO45. We describe steps for preparing cel...

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Detalles Bibliográficos
Autores principales: Lin, Xiao-Tong, Zhang, Jie, Xie, Chuan-Ming
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10050764/
https://www.ncbi.nlm.nih.gov/pubmed/36964909
http://dx.doi.org/10.1016/j.xpro.2023.102199
Descripción
Sumario:E3 ubiquitin ligases play a role in protein degradation, cellular localization, and activation, and their dysregulation is associated with human diseases. Here, we present a protocol to detect IGF2BP1 ubiquitination and activation by an E3 ubiquitin ligase FBXO45. We describe steps for preparing cells and transfecting plasmids. We detail the use of western blot to detect IGF2BP1 ubiquitination and a Cell Counting Kit-8 (CCK-8) assay to detect IGF2BP1 activation. This protocol is applicable to other proteins of interest. For complete details on the use and execution of this protocol, please refer to Lin et al. (2021).(1)

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