FIGURE 3.

Effect of suppression of expression of NDUFAF5 on hydroxylation of Arg-73 in NDUFS7. Part A, HEK293T cells overexpressing tagged-NDUFS7 were transfected twice at 0 and 72 h, with 50 or 100 nm siRNA specific for NDUFAF5 (-FAF5), NDUFAF7 (-FAF7), or with a negative control siRNA. Transcript levels were examined at 48 and 120 h and are normalized to endogenous β-actin. The error bars show the standard deviation. Part B, mass spectrometric analysis of the hydroxylation of Arg-73 in NDUFS7. The histograms are derived from the extracted ion chromatograms for the m/z values of the hydroxylated or non-hydroxylated Asp-N peptide (residues 66–79) from NDUFS7. The numbers correspond to the level of hydroxylation relative to a value of 1.0 for the untreated control. Parts C and D, fragmentation spectra of triply charged ions, m/z 541.29 and m/z 546.62, representing unmodified and hydroxylated versions of the AspN peptide (resides 66–79) from human NDUFS7. In the insets, fragment ions are mapped onto the amino acid sequence.