Abstract
The detection of protein–protein interactions by imaging techniques often requires the overexpression of the proteins of interest tagged with fluorescent molecules, which can affect their biological properties and, subsequently, flaw experiment interpretations. The recent development of the proximity ligation assays (PLA) technology allows easy visualization of endogenous protein–protein interactions at the single molecule level. PLA relies on the use of combinations of antibodies coupled to complementary oligonucleotides that are amplified and revealed with a fluorescent probe, each spot representing a single protein–protein interaction. Another application of this technique is the detection of proteins posttranslational modifications to monitor their localization and dynamics in situ. Here, we describe the use of PLA to detect protein SUMOylation, a posttranslational modification related to ubiquitination, as well as interaction of SUMOylated substrates with other proteins, using both adherent and suspension cells.
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Acknowledgments
MP’s laboratory is an “Equipe Labellisée” of the Ligue Nationale contre le Cancer. This work was also supported by the CNRS, the Association pour la Recherche sur le Cancer (ARC), the INCA (PLBIO 2012-105 and 2013-1-MELA-05-1), the Marie-Curie Initial Training Network “UPStream,” and the Region Languedoc Roussillon (programme “Chercheur d’Avenir”). The authors would like to acknowledge networking support by the Proteostasis COST Action (BM1307).
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Ristic, M., Brockly, F., Piechaczyk, M., Bossis, G. (2016). Detection of Protein–Protein Interactions and Posttranslational Modifications Using the Proximity Ligation Assay: Application to the Study of the SUMO Pathway. In: Matthiesen, R. (eds) Proteostasis. Methods in Molecular Biology, vol 1449. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3756-1_17
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DOI: https://doi.org/10.1007/978-1-4939-3756-1_17
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