CAMKIIα directly phosphorylates Nox5 on Ser498 and Ser494 in an in vitro kinase assay. A, Nox5 was immunoprecipitated from COS-7 cells transduced with HA-Nox5 adenovirus and subject to an in vitro kinase assay. Phosphorylated samples were immunoblotted for phosphorylated Ser490, Thr494, and Ser498 versus total Nox5. COS-7 cells were cotransfected with either T494A (B), S498A (C), or S490A, T494A, and S498A (D) triple mutants of HA-Nox5 together with WT CAMKIIα or control DNA (lacZ). Basal superoxide was measured by L-012. Relative expression of Nox5 was detected by immunoblotting (bottom). Results are presented as mean ± S.E.M. (n = 4–6); *, p < 0.05 versus control (lacZ).