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

Cellular Src tyrosine kinase (c-Src) exists in the secretomes of several human can-cers (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 tyro-sine-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 exam-ining 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 Sanchez-Pozo et al. (2018).2