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CD95 pp 41-48 | Cite as

Proximity Ligation Assay (PLA) to Evaluate DISC and MISC Composition

  • Melissa Thomas
  • Patrick Legembre
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1557)

Abstract

Distribution of the death receptor CD95 into lipid rafts (aggregation) and/or its internalization may contribute to the implementation of the apoptotic signal at the detriment of the non-apoptotic signaling pathway [1–6]. Also CD95 can form different protein complexes via dynamic protein–protein interactions (PPIs) according to its interaction with soluble or transmembrane CD95L. Therefore, spatiotemporal identification of these PPIs is pivotal to anticipate the signaling pathway implemented in cells stimulated with different forms of CD95L. Also, many disorders result from dysfunctions in terms of PPI subcellular distribution and/or their intensity, rendering evaluation of these features crucial to better understand pathogenesis.

In situ proximity ligation assay (PLA) is a methodology that offers the possibility to identify PPIs and to determine where these PPIs occur in subcellular location (Fig. 1). Moreover, based on imaging, this method allows a quantification of PPIs at the cellular level and with a higher specificity than classical immunofluorescence assays. We here describe PLA used to confirm CD95/FADD interaction, a protocol that may serve to highlight other CD95 partners.

Key words

CD95 Death inducing signaling complex FADD Protein–protein interaction Proximity ligation assay 

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Centre Eugène MarquisRennesFrance
  2. 2.INSERM ERL440-OSSEquipe Labellisée Ligue Contre Le CancerRennesFrance
  3. 3.Université de Rennes-1RennesFrance

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