Characterization of ATG8-family protein phosphorylation by Phos-tag gel for autophagy study.
STAR Protoc
; 5(1): 102872, 2024 Mar 15.
Article
em En
| MEDLINE
| ID: mdl-38319785
ABSTRACT
Autophagy supports cell survival under different stress conditions, where ATG8-family proteins are required for autophagosome biogenesis/maturation and selective autophagy. Here, we present a protocol for studying ATG8-family protein phosphorylation using Phos-tag gel, a modified SDS-PAGE system, when the related phosphorylation site information and/or specific phospho-antibody are unavailable. We describe steps for generating GST-ATG8 proteins in bacteria, purifying S protein-Flag-SBP protein (SFB)-tagged kinasefrom cells, preparing gel, and an in vitro kinase assay. We then detail procedures for western blotting and image processing. For complete details on the use and execution of this protocol, please refer to Seo et al.1.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Piridinas
/
Autofagia
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article