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Flow cytometric in situ proximity ligation analyses of protein interactions and post‐translational modification of the epidermal growth factor receptor family

Interactions between members of the epidermal growth factor receptor (EGFR) family mediates cellular responses to ligand stimulation. Measurement of these interactions could provide important information and may prove useful as prognostic markers in malignancy. Therefore, to develop methods to study these interactions in genetically unmodified cells, such as clinical samples, in a sensitive and selective way, with good statistical accuracy, is important. The in situ proximity ligation assay (in situ PLA) was used to quantify homo‐ and heteromeric interactions between EGFR and HER2 in cultured cells, using flow cytometry as the readout method. Cells were monitored for changes in dimerization patterns and phosphorylation status upon stimulation. The different cell lines displayed varying amounts of interactions between EGFR and HER2, but the amount of dimerization was not found to be affected significantly upon stimulation by EGF. Activation of EGFR could be visualized by in situ PLA, but not by immunofluorescence staining. In situ PLA was successfully used to study receptor dimerization and activation of the EGF‐receptor family with high selectivity and sensitivity. The combination of in situ PLA and flow cytometry provided a statistically powerful way of analyzing protein‐protein interactions and post‐translational modifications on a single‐cell basis. © 2009 International Society for Advancement of Cytometry