Journal: Journal of Molecular Signaling
Article Title: NADPH oxidase mediates the oxygen-glucose deprivation/reperfusion-induced increase in the tyrosine phosphorylation of the N -methyl-D-aspartate receptor NR2A subunit in retinoic acid differentiated SH-SY5Y Cells
doi: 10.1186/1750-2187-7-15
Figure Lengend Snippet: Increased ROS generation during reperfusion of OGD subjected cultures of retinoic acid differentiated SH-SY5Y involves NADPH oxidase activity. Representative fluorescent images from three independent experiments of reactive oxygen species production following OGD ( A ) with vehicle (1:1000 DMSO) or DPI (100 nM) detected using dihydroethidium (DHE). Spectrophotometric quantification of reactive oxygen species following OGD ( B ) with vehicle (1:1000 DMSO) or DPI (100 nM) utilizing nitrobluetetrazolium chloride (NBT). ( C ) Western blot shown is representative of three independent experiments comparing p67 phox , NR2A subunit protein expression in non- and differentiated SH-SY5Y cells. ( D ) Quantification of the relative densitometry of p67 phox immunoreactive band in non- and differentiated SH-SY5Y cells. Data represent fold change of OGD/R groups versus normoxic control group (arbitrary units) ± S.E.M from three separate experiments that consisted of at least 6 determinents (askteriks * indicates a p <0.05 from vehicle treated normoxic control; ANOVA with post hoc Bonferroni test). Phorbol 12-myristate 13-acetate (PMA; 1 μM for 15 minutes) was used as a positive control for NADPH oxidase activity in both the DHE and NBT assays. Quantitative data from p67 phox protein expression represent fold change of differentiated SH-SY5Y compared to non-differentiated SH-SY5Y cells (arbitrary units) ± S.E.M from three separate experiments (askteriks * indicates a p <0.01 from non-differentiated control; student t -Test).
Article Snippet: The affinity purified rabbit-polyclonal p67 phox antibody (1:1000) was purchased from Millipore (Billerica, MA, USA).
Techniques: Activity Assay, Western Blot, Expressing, Control, Positive Control
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