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NADPH Oxidases pp 447-458 | Cite as

Protein–Protein Interaction Assay to Analyze NOX4/p22phox Heterodimerization

  • Sharon O’Neill
  • Ulla G. KnausEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1982)

Abstract

The stabilization and activation of NOX4 through its binding with p22phox are well documented; however little is known of the precise manner by which these two proteins interact. In recent years, the field of proteomics has undergone tremendous development with the introduction of many novel methods for the identification and characterization of protein–protein interactions (PPIs). To enhance our understanding of structural determinants leading to the association between NOX4 and p22phox, we developed a binary luciferase reporter assay (NanoBiT®) to quantitatively assess NOX4-p22phox heterodimerization. The complementation reporter quantitatively determines the accurate, reduced, or failed complex assembly, which can be confirmed and further interrogated by analyzing NOX4 catalytic activity (H2O2 release), protein expression, and dimer localization. This association-based PPI technique represents both a much-needed expansion of the NOX4 lead discovery tool box and a versatile method to probe the architecture of NOX and DUOX complexes in the future.

Key words

NADPH oxidase NOX4 p22phox Protein–protein interaction (PPI) Heterodimerization NanoBiT® Bioluminescence 

Notes

Acknowledgments

This work was supported by Science Foundation Ireland (UGK) and the MolCellBiol Programme (Programme for Research in Third-Level Institutions, co-funded under the EU Regional Development Fund) (SON). We thank M. Mathis and R. Bouhelal of the Novartis Institutes for Biomedical Research, Switzerland, and Promega Corporation, Wisconsin, USA, for the collaborative effort in establishing this assay and for providing reagents.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Conway Institute, School of MedicineUniversity College DublinDublinIreland

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