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Guidelines for the Detection of NADPH Oxidases by Immunoblot and RT-qPCR

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NADPH Oxidases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1982))

Abstract

The identification of NADPH oxidase (NOX) isoforms in tissues is essential for interpreting experiments and for next step decisions regarding cell lines, animal models, and targeted drug design. Two basic methods, immunoblotting and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), are important to monitor NOX protein and messenger RNA (mRNA) levels, respectively, for a range of investigations from understanding cell signaling events to judging NOX inhibitor efficacies. For many other genes that are expressed in high abundance, these methods may seem rather simple. However, detecting the low expression levels of endogenous NOX/DUOX is difficult and can be frustrating, so some guidelines would be helpful to those who are facing difficulties. One reason why detection is so difficult is the limited availability of vetted NOX/DUOX antibodies. Many of the commercial antibodies do not perform well in our hands, and dependable antibodies, often generated by academic laboratories, are in limited supply. Another problem is the growing trend in the NOX literature to omit end-user validation of antibodies by not providing appropriate positive and negative controls. With regard to NOX mRNA levels, knockdown of NOX/DUOX has been reported in cell lines with very low endogenous expression (C q values ≥30) or in cell lines devoid of the targeted NOX isoform (e.g., NOX4 expression in NCI-60 cancer cell panel cell line 786–0). These publications propagate misinformation and hinder progress in understanding NOX/DUOX function. This chapter provides overdue guidelines on how to validate a NOX antibody and provides general methodologies to prepare samples for optimal detection. It also includes validated methodology to perform RT-qPCR for the measurement of NOX mRNA levels, and we suggest that RT-qPCR should be performed prior to embarking on NOX protein detection.

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Acknowledgments

We thank for their generous support of this work: Dr. Ajay Shah and Dr. Pidder Jansen-Dürr for the NOX4 polyclonal rabbit antibodies; Dr. William Kaelin for the RCC4 parent cell line; Dr. Ralf Brandes for HEK 293 cells stably expressing NOX4 or NOX1; and Dr. Vincent Jaquet, Dr. Mark Quinn, Dr. Corinne Dupuy, Dr. Chihiro Yabe-Nishimura, Dr. Misaki Matsumoto, Dr. Ulla Knaus, and Dr. William Nauseef for contributing specific information for some of the NOX/DUOX antibodies and reviewing the manuscript. We also thank the editors, Dr. Ulla Knaus and Dr. Tom Leto, for the invitation to contribute a chapter to this book.

This chapter is dedicated to the late Dr. Gary Bokoch (The Scripps Research Institute, La Jolla, CA, USA), who made many contributions to the field of NADPH oxidases and served as a post-doctoral mentor of both Dr. Becky Diebold and Dr. Ulla Knaus.

This work was made possible by funding from the National Institute of Health, USA, and the American Heart Association. XDD is supported by the “Fonds de la Recherche Scientifique” (FRS-FNRS), the “Fonds Docteur J.P. Naets” managed by the “Fondation Roi Baudouin,” and the “Fondation Tournay-Solvay.”

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Authors and Affiliations

  1. Department of Pathology, Emory University, Atlanta, GA, USA

    Becky A. Diebold, S. Garrett Wilder, James W. McCoy, Yerun Zhu & J. David Lambeth

  2. Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles (ULB), Brussels, Belgium

    Xavier De Deken

  3. Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

    Jennifer L. Meitzler & James H. Doroshow

  4. Division of Cancer Treatment and Diagnosis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

    James H. Doroshow

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  1. Becky A. Diebold
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  2. S. Garrett Wilder
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  3. Xavier De Deken
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  4. Jennifer L. Meitzler
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  5. James H. Doroshow
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  6. James W. McCoy
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  7. Yerun Zhu
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  8. J. David Lambeth
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Corresponding author

Correspondence to Becky A. Diebold .

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Editors and Affiliations

  1. Conway Institute, School of Medicine, University College Dublin, Dublin, Ireland

    Ulla G. Knaus

  2. Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Thomas L. Leto

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Diebold, B.A. et al. (2019). Guidelines for the Detection of NADPH Oxidases by Immunoblot and RT-qPCR. In: Knaus, U., Leto, T. (eds) NADPH Oxidases. Methods in Molecular Biology, vol 1982. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9424-3_12

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  • DOI: https://doi.org/10.1007/978-1-4939-9424-3_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9423-6

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