Fig. 4.
12-Lipoxygenase (12-LOX) activation in neurons following 15-A2t-isoprostane (15-A2t-IsoP) exposure causes cell death. Neurons were exposed to (a) vehicle or (b) 30 μM 15-A2t-IsoP for 3 h then fixed, stained with an anti-12-LOX antibody, and subjected to fluorescent microscopy. Translocation of 12-LOX to the membrane is associated with its activation (arrowheads). Photomicrographs are representative of results from three independent experiments. (c) Immature neurons were exposed to vehicle (Con), 30 μM 15-A2t-IsoP + baicalein (Baic: 20 μM) or 5 mM glutamate (Glut, used as a positive control) for 3 h, then membrane and cytoplasmic fractions were prepared and analyzed for 12-LOX by western blot. Blots are representative of two independent experiments. (d) Neurons were exposed to 15-A2t-IsoP (30 μM) + the specific 12-LOX inhibitor baicalein (Baic: 20 or 2 μM). After 20–24 h, cell death was assessed by lactate dehydrogenase (LDH) assay. Baicalein was present for 30 min before and during IsoP exposure. Data represents the mean + SEM for at least three independent experiments, and was analyzed by one-way ANOVA. *p < 0.05 vs. IsoP alone.