Figure 2.

Upregulation and activation of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase (NOX) after transient global cerebral ischemia (tGCI). (A) Western blots using whole cell lysate samples show biphasic upregulation of the cytosolic subunits (p47^phox and p67^phox) at the early (3 to 6 hours) and late (72 hours) phases after tGCI. In contrast, the membrane-bound subunit gp91^phox upregulated 24 and 72 hours after tGCI (n=4, ^*P<0.05 compared with sham). S, sham; OD, optical density. (B) Western blot analysis showed increase of cytosolic subunits (p47^phox and p67^phox) in the membrane fraction 6 and 72 hours after ischemia (n=5, ^*P<0.05 compared with sham). In the cytosolic fraction, no significant differences were seen at any time point. β-Actin and Na-K ATPase were used as internal controls for the cytosolic and membrane fractions, respectively. (C) Representative confocal images of immunostaining. Expression of p47^phox became more intense, especially on the cell surface (arrowheads), 6 hours after ischemia. Microtubule-associated protein-2 (MAP2) immunostaining demarcates the neuronal cytoplasmic space. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar=20 μm.