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Proximity ligation assay (PLA) is a method that allows the identification of epitopes located in the immediate vicinity (distance <40 nm) of each other, in cultured cells or tissue samples. It is used for the in situ detection of protein–protein interactions or to detect and quantify protein modifications with high sensitivity. The locations of proximate epitopes appear as dots detected by confocal microscopy. In this chapter, we describe an optimized protocol in endothelial cells that can be used to identify protein–protein interactions (e.g., direct interaction of ανβ3 integrin with neuropilin-1) or protein phosphorylation (e.g., phosphorylation of ribosomal protein S6 kinase B1 (S6K1)), which is a biomarker for mTORC1 activation. Representative confocal microscope photos are presented, and methods to quantify the results are discussed.

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Identifying Protein–Protein Interaction or Protein Phosphorylation by Proximity Ligation Assay (PLA) in Endothelial Cells

  • 实验方案

  • 发表日期：

    2025年

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  • Eleni Mourkogianni,
  • Marina Koutsioumpa,
  • Evangelia Papadimitriou

摘要

Proximity ligation assay (PLA) is a method that allows the identification of epitopes located in the immediate vicinity (distance <40 nm) of each other, in cultured cells or tissue samples. It is used for the in situ detection of protein–protein interactions or to detect and quantify protein modifications with high sensitivity. The locations of proximate epitopes appear as dots detected by confocal microscopy. In this chapter, we describe an optimized protocol in endothelial cells that can be used to identify protein–protein interactions (e.g., direct interaction of ανβ3 integrin with neuropilin-1) or protein phosphorylation (e.g., phosphorylation of ribosomal protein S6 kinase B1 (S6K1)), which is a biomarker for mTORC1 activation. Representative confocal microscope photos are presented, and methods to quantify the results are discussed.