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

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.

NADPH Oxidase 3 Deficiency Protects From Noise-Induced Sensorineural Hearing Loss

“…To palliate the lack of specific antibodies against Nox3 ( Diebold et al, 2019 ), Mohri et al recently developed a mouse model with a reporter gene expressed upon Nox3 promoter ( Mohri et al, 2021 ). In situ hybridization is another sensitive and reliable method to detect NOX mRNA ( Moll et al, 2018 ; Rousset et al, 2020 ).…”

Transcriptomics of single dose and repeated carbon black and ozone inhalation co-exposure highlight progressive pulmonary mitochondrial dysfunction

“…NADPH oxidase system is among the best described pathways connecting inflammatory response and mitochondrial dysfunction through reactive oxygen species (ROS) production [ 81 – 85 ]. Real time PCR has recently been recommended as the method of choice for initial screening of NADPH pathway [ 86 ]. Our results clearly indicated increased mRNA expression of Nox2 as well as Duox2 subunits as well as adaptor proteins P40 phox , and P47 phox .…”

Di-Tyrosine Crosslinking and NOX4 Expression as Oxidative Pathological Markers in the Lungs of Patients with Idiopathic Pulmonary Fibrosis

“…This antibody belongs to the small list of available validated NOX4 antibodies, suitable for Western blot and IHC [ 21 ]. We previously documented the specificity of this antibody over other NOX isoforms [ 22 ] and its specificity for immunohistochemical detection in human tissue samples [ 23 ]. The mouse monoclonal DT antibody was generated using a DT conjugate as immunogen and selected for DT specificity [ 24 ].…”