Fig. 4

The physical acetylation of MnSOD-K68 produces peroxidase activity. a–d Immortalized MnSOD^−/− MEFs expressing Flag-MnSOD^WT were cultured in NAM + TSA or NAD + , separated using a 50 kDa molecular cutoff membrane and a MnSOD-K68-Ac, MnSOD, and actin immunoreactive protein levels were determined. b Peroxidase activity and c MnSOD activity in < 50 kDa fractions. d The > 50 kDa fractions were analyzed for MnSOD activity. e Bacterially produced and purified recombinant MnSOD-WT and MnSOD-K68-Ac proteins were characterized by size exclusion column chromatography. Standards are shown. f, g Elution volumes 13 and 14 mL, corresponding to peak 1/blue (f) and elution volumes 16 and 17 mL, corresponding to peak 2/red (g) from Fig. 4e were analyzed for MnSOD by MnSOD-K68-Ac immunoblotting (top panels) or Coomassie Brilliant Blue staining (bottom panels). h, i Peak 1 (elution volumes 13 and 14 mL) and peak 2 (elution volumes 16 and 17 mL) were analyzed for h superoxide dismutase activity i and peroxidase activity. All experiments were done in triplicate. Errors represent ± 1 SEM. ***p < 0.01. A t-test was used to compare means of the two groups