Figure 6. SH-SY5Y human neuroblastoma cells express a functional NADPH oxidase activity.

(A) Whole cell lysates of human SH-SY5Y neuroblastoma cells revealed immunoreactivity against both membrane-bound subunits (gp91^phox, p22^phox) and the cytosolic subunits (p67^phox, p47^phox, and p40^phox) of the NOX2 complex using anti-human subunit specific antibodies (loading-control: actin). Double bands were routinely detected for both p67^phox and p47^phox. (B) Treatment of SH-SY5Y cells over a 60 min time course with 200 nM PMA and 50 μM AA stimulated translocation of p67^phox into plasma membrane (arrowhead). Equal amounts of total plasma membrane protein were separated by SDS-gel electrophoresis followed by western blotting against p67^phox. (C) SH-SY5Y cells were incubated with 100 ng/ml TNFα for 15 min. Plasma membrane proteins were biotinylated with Sulfo-NHS-biotin and affinity-purified on streptavidin-agarose. Equal amounts of membrane protein were separated by SDS-gel electrophoresis followed by western blotting, and immunoreactivity against p67^phox was quantified (chemiluminescence). TNFα elicited a significant translocation of p67^phox to plasma membranes (black bar, TNFα) demonstrating NOX activity, which was blunted by 10 μM ATK (grey bar, TNF+ATK) to levels similar of control (open bar, CON). (D) SH-SY5Y cells were incubated with TNFα (100 ng/ml, 15 min) and then lysed. Whole cell lysates were subjected to gel electrophoresis followed by western blotting and quantification of phospho-p40^phox immunoreactivity (chemiluminescence). TNFα significantly increased levels of phospho-p40^phox (black bar, TNFα) in SH-SY5Y cells compared to control (open bars, CON) indicative of NOX activation. All values were normalized to control and data represent the mean of three independent experiments ± standard deviations (*p<0.05)