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. 2023 Dec 15;4(4):102755.
doi: 10.1016/j.xpro.2023.102755. Epub 2023 Dec 2.

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

Michael P Omini  1 , Deema Alsalih  2 , SarahBeth D Votra  2 , Dimitra Bourboulia  3
Affiliations

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

Michael P Omini et al. STAR Protoc. .

Abstract

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.

Keywords: Cancer; Cell Biology; Molecular Biology.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Western Blotting of total protein extracts from SYF and SYF+c-Src cells Probing for c-Src and total input GAPDH.
Figure 2
Figure 2
Schematic of protein secretion pathways Conventional (ER/Golgi) and non-conventional (multivesicular bodies/MVB) pathways.
Figure 3
Figure 3
Secretomes and downstream applications
Figure 4
Figure 4
Concentrating cell-conditioned media (CM) using a 4 mL - 10K cutoff unit (A) One Amicon Ultra Centrifugal Filter 10K-cutoff unit. (B) 4 mL CM were added into the concentrator. (C) CM is centrifuged at 3,220 g and at 4°C. (D) After concentration, top is the concentrated CM and bottom is the flowthrough.
Figure 5
Figure 5
Coomassie blue dye staining SDS-PAGE gel showing total protein loading from concentrated 10X CM with indicated treatments.
See this image and copyright information in PMC

References

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