Response to Pagano et al.

In their letter, Pagano et al. appreciate the development of the Nox1, Nox2, and Nox4 triple (3N(-/-)) knockout mouse. They also agree on the view that chemiluminescence assays in general have severe limitations. However, they criticize the fact that the membrane assays in the particular study were restricted to chemiluminescence techniques. Moreover, Pagano et al. got the impression that statements concerning membrane assays of Nox activity in general were made. In addition to a lack of some technical details, Pagano et al. also found the characterization of the 3N(-/-) incomplete and some of the results to be incomprehensible. Although we are grateful for the interest of Pagano et al. in our work, we realized that basically each observation of our study was questioned. This is certainly an excessive rejection of the study in total and fails to appreciate the clear chain of evidences presented. Our work focused on chemiluminescence, and thus, any conclusions are restricted to this technique. Moreover, the 3N(-/-) mice were never developed to study the physiology of Nox enzymes, but rather to validate Nox specificity of NADPH-stimulated chemiluminescence assays. We are convinced that our findings are a valid demonstration that chemiluminescence-based assays in membrane preparations stimulated with NADPH do not measure Nox activity. This conclusion is based on both overexpression studies as well as genetic deficient mouse models. The criticisms of Pagano et al. thus might be justified in some aspects; they, however, cannot disprove the conclusions of our work. Antioxid. Redox Signal. 23, 1247-1249.