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Endothelial cells express constitutively a Nox2 oxidase, which by generating reactive oxygen species (ROS) plays an important role in TNFα signalling. The Nox2 has at least four regulatory subunits and p47phox is a major regulatory subunit of this enzyme. It has been reported that phosphorylation of double serines (S303/S304) in p47phox is a key step for Nox2 activation. In this study, we investigated the role of p47phox S303/S304 phosphorylation in TNFα-induced ROS production and mitogen-activated protein kinase (MAPK) activation in endothelial cells. Serines 303/304 (human p47phox cDNA) were replaced to alanines by site-directed mutagenesis and the wild-type and mutated p47phox were used to transfect a mouse microvascular endothelial cell line (SVEC4-10). Forty-eight hours after transfection, cells were stimulated with or without TNFα (100 U/ml for 30 minutes) and examined for ROS production (lucigenin emiluminescence) and MAPK activation (Western blot). Compared with cells transfected with an empty vector control) or cells transfected with wild-type p47phox (overexpression), S303/304A mutation had no significant effect on the basal level (without TNFα) of ROS production. When cells were stimulated with TNFα, there was a twofold increase in ROS production by control and p47phox overexpressed cells. TNFα-induced ROS production was significantly reduced (∼60%) in cells transfected with the S303/304A mutation. However, there was no significant difference in TNFα-induced phosphorylation of ERK1/2, p38MAPK and JNK between controls and the cells transfected with the S303/304A mutation. In conclusion, p47phox phosphorylation at S303/S304 plays an important role in TNFα-induced ROS production, but is not involved in TNFα-induced MAPK activation.