CAMKII-dependent phosphorylation of Nox5 on Ser475 in intact cells. A, COS-7 cells were cotransfected with WT Nox5 and S475A Nox5 together with CAMKIIα, and cell lysates were probed for the relative expression of total HA-Nox5. B, phosphoproteins in cell lysates were bound to phosphoprotein affinity columns, and eluted proteins were probed for the expression of HA-Nox5. C, detection of phosphorylated amino acid modifications of Nox5 in COS-7 cells cotransfected with Nox5 and CAMKIIα. MS/MS spectra of parent ion m/z 1822.7 was obtained by MALDI-TOF-MS/MS fitted with peptide 464-SQGQWTNRLYES*FK-477 from the Nox5β sequence phosphorylated at Ser475.