Immunoprecipitation and Western blot-based detection of protein O-GlcNAcylation in cells

Detection of protein O-GlcNAcylation could be challenging. By using the host-cell factor 1 (HCF-1), a known O-GlcNAcylated protein, we immunoprecipitated HCF-1 from transfected HEK293T cells or endogenous HCF-1 from HeLa cells to detect its O-GlcNAc levels by Western blotting. We also take advantage...

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Detalles Bibliográficos
Autores principales: Ahmed, Oumaima, Affar, Malik, Masclef, Louis, Echbicheb, Mohamed, Gushul-Leclaire, Mila, Estavoyer, Benjamin, Vocadlo, David J., Affar, El Bachir
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8784402/
https://www.ncbi.nlm.nih.gov/pubmed/35106498
http://dx.doi.org/10.1016/j.xpro.2021.101108
Descripción
Sumario:Detection of protein O-GlcNAcylation could be challenging. By using the host-cell factor 1 (HCF-1), a known O-GlcNAcylated protein, we immunoprecipitated HCF-1 from transfected HEK293T cells or endogenous HCF-1 from HeLa cells to detect its O-GlcNAc levels by Western blotting. We also take advantage of RNAi or chemical inhibitors to modulate OGT and OGA activities before HCF-1 immunoprecipitation. For complete details on the use and execution of this protocol, please refer to Daou et al. (2011).

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