A Nonpolar Blueberry Fraction Blunts NADPH Oxidase Activation in Neuronal Cells Exposed to Tumor Necrosis Factor-α
Figure 5
Nonpolar blueberry fraction inhibits association of p67phox into plasma membranes without disrupting phosphorylation of p40phox. Serum free cultures of SH-SY5Y cells were incubated with nonpolar (NPBB) and polar (POBB) blueberry fractions (5 μg/mL each, 1 h) prior to insult with 200 ng/mL TNFα or 400 ng/mL PMA (30 min). (a) After cell lysis and subcellular fractionation, equal amounts of total plasma membrane protein were subjected to SDS gel electrophoresis followed by western blotting, immunoreactivity against p67phox (colorimetric detection) was quantified by densitometry (ImageJ64), and all values were normalized to control. TNFα and PMA (filled bars, resp.) induced significant association of p67phox in plasma membrane fractions compared to control (open bar). Preincubation of SH-SY5Y cells with NPBB prevented p67phox plasma membrane association regardless of stimulus (TNFα or PMA) whereas preincubation with POBB was ineffective. Note that incubation of SH-SY5Y cells with POBB alone but not NPBB resulted in a significant increase in p67phox plasma membrane association. (b) Immunoreactivity against phosphorylated p40phox was determined in whole cell lysates (ELISA). All data was adjusted total cellular protein (BCA assay) and normalized to controls. Exposure to 400 ng/mL PMA significantly stimulated phosphorylation of p40phox (solid bar), necessary for NOX activity, compared to control (CON, open bar). Importantly, pretreatment of SH-SY5Y cells with 5 μg/mL NPBB had no effect on the extent of p40phox phosphorylation (grey bar). All data represent the mean of at least four independent experiments ± standard error of the mean (SEM), and statistical significance was determined at * (ANOVA and Tukey’s post hoc analysis).
