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. 2012 Mar 13;2012:768101. doi: 10.1155/2012/768101

Figure 4.

Figure 4

Nonpolar blueberry fractions block superoxide production and abolish p67phox accumulation at plasma membranes in response to TNFα. Serum free cultures of SH-SY5Y were incubated with pharmacological inhibitors or blueberry fractions (NPBB or POBB; 5 μg/mL each, 1 h) prior to TNFα (200 ng/mL) or PMA (400 ng/mL) exposure (30 min) and subjected either to a cytochrome C reduction assay or an ELISA targeting plasma membrane associated p67phox. (a) Cytochrome C reduction was assayed in whole cell lysates. TNFα exposure of SH-SY5Y cells caused an increase in superoxide production (black bar) as indicated by its sensitivity to a presence of 200 U/mL SOD (hatched bars in all conditions) compared to control (open bar). Preincubation of SH-SY5Y cells with 10 μM DPI, 2 mM AE (AEBSF), or 2 mM NAC all inhibited TNFα-mediated superoxide production (light grey bars). Also, pretreatment of SH-SY5Y cells with nonpolar blueberry fractions (NPBB) negated superoxide production upon exposure to TNFα (dark grey bars) as suggested by SOD sensitivity. In contrast, pretreatment of SH-SY5Y cells with polar blueberry fractions (POBB) was ineffective in abolished TNFα-mediated superoxide production. All data was normalized to control and represent the mean of two independent experiments ± range. (b and c) Plasma membrane-associated p67phox was quantified (20 μg/mL total membrane protein) using an ELISA (see Section 2) after sucrose gradient centrifugation of cell lysates to obtain a plasma membrane fraction. (b) TNFα exposure of SH-SY5Y cells significantly increases plasma membrane-association of p67phox (filled bar) compared to control (open bar). Preincubation of SH-SY5Y cells with NPBB completely blocked an association of p67phox with plasma membranes upon addition of TNFα whereas preincubation with POBB was ineffective (grey bars). (c) PMA also stimulated an increase in p67phox association with plasma membranes (filled bar) in SH-SY5Y cells compared to control (open bar). As in the case of TNFα, preincubation of SH-SY5Y cells with NPBB successfully negated an association of p67phox with plasma membranes (grey bars) compared to control (open bar). Preincubation of SH-SY5Y cells with POBB did not negate PMA-mediated p67phox plasma membrane association. All values were normalized to control, data represent the mean of at least four independent experiments ± standard deviations, and statistical significance was determined at *P < 0.05 (ANOVA and Tukey's post hoc analysis).