Fig 7.

Monensin elevated the mitochondrial MnSOD protein level without a significant effect on the cytosolic MnSOD protein abundance (A, n = 5). Ouabain (4 nM) inhibited the effect of monensin on the mitochondrial MnSOD protein (B, n = 5). Membranes were probed with antibodies against aldose reductase (AR, cytosolic marker) and cytochrome c oxidase subunit IV (COX4, mitochondrial marker) to show adequate separation of these two compartments and with actin antibody to show roughly equal loading. Monensin had no significant effect on the total activity of cytosolic or mitochondrial MnSOD (C, n = 5), but significantly reduced the specific activity of mitochondrial MnSOD and this effect was blocked by ouabain (D, n = 5). HEK293 cells were treated as in Fig 5. The total and specific MnSOD activities were measured as in Fig 4, but the total and specific activities were normalized to the control in each experiment. (*p < 0.05 vs Control or Monensin plus Ouabain in D, **p < 0.05 vs Monensin, One-way ANOVA for A, Two-way ANOVA for B and D).