Protocol to characterize extracellular c-Src tyrosine kinase function through substrate interaction and phosphorylation

Autor: Michael P. Omini, Deema Alsalih, SarahBeth D. Votra, Dimitra Bourboulia
Jazyk: angličtina
Rok vydání: 2023
Předmět:
Zdroj: STAR Protocols, Vol 4, Iss 4, Pp 102755- (2023)
Druh dokumentu: article
ISSN: 2666-1667
DOI: 10.1016/j.xpro.2023.102755
Popis: Summary: Cellular Src tyrosine kinase (c-Src) exists in the secretomes of several human cancers (extracellular, e-Src). Phosphoproteomics has demonstrated the existence of 114 potential extracellular e-Src substrates in addition to Tissue Inhibitor of Metalloproteinases 2. Here, we present a protocol to characterize secreted tyrosine-phosphorylated substrates as a result of c-Src expression and secretion. We describe steps for collecting cell secretomes and extracts, performing antibody treatment and Ni-NTA pull-down, and detecting protein-protein interaction and substrate Y-phosphorylation. This protocol is adaptable for studies examining the function of other extracellular kinases.For complete details on the use and execution of this protocol, please refer to Backe et al. (2023)1 and Sánchez-Pozo et al. (2018).2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
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