PDF(Sci-hub)
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.
摘要：
组织中NADPH氧化酶（NOX）亚型的鉴定对于解释实验和关于细胞系的下一步决策至关重要。动物模型,和靶向药物设计。两种基本方法，免疫印迹和逆转录酶定量聚合酶链反应（RT-qPCR），对监测NOX蛋白和信使RNA（mRNA）水平很重要，分别,从了解细胞信号传导事件到判断NOX抑制剂疗效的一系列研究。对于许多其他高丰度表达的基因，这些方法可能看起来相当简单。然而,检测内源性NOX/DUOX的低表达水平是困难的，所以一些指导方针会对那些面临困难的人有所帮助。检测如此困难的一个原因是经过审查的NOX/DUOX抗体的可用性有限。许多商业抗体在我们手中表现不佳，和可靠的抗体，通常由学术实验室产生，供应有限。另一个问题是NOX文献中通过不提供适当的阳性和阴性对照而忽略抗体的最终用户验证的增长趋势。关于NOXmRNA水平，在内源性表达非常低（Cq值≥30）的细胞系或缺乏靶向NOX同种型的细胞系中，已经报道了NOX/DUOX的敲低（例如，NCI-60癌细胞组细胞系786-0中的NOX4表达)。这些出版物传播了错误信息，阻碍了了解NOX/DUOX功能的进展。本章提供了有关如何验证NOX抗体的过期指南，并提供了准备样品以进行最佳检测的一般方法。它还包括经过验证的方法来进行RT-qPCR以测量NOXmRNA水平，我们建议在开始NOX蛋白检测之前进行RT-qPCR。
