Fig. 5. Oxidative insults induce formation of HNE–protein conjugates in neural cells: attenuation by GSH and Bcl-2.A, Hippocampal cultures were exposed for 2 hr to 0.2% ethanol (Control), 2 μmFeSO4, 2 μmHNE, or 1 mm GSH plus 2 μm HNE (GSH+HNE). Then cells were fixed and immunostained with HNE antibody. Arrowheads point to neuronal cell bodies.B, PC12-V cells and PC12-Bcl-2 cells were exposed for 2 hr to vehicle (0.2% ethanol) or 10 μm HNE. Then cells were fixed and immunostained with anti-HNE antibody. Note the much greater level of HNE immunoreactivity in PC12-V cells exposed to HNE, as compared with PC12-Bcl-2 cells exposed to HNE. C, Cell homogenates from untreated control PC12-V cultures (c), PC12-V cultures exposed to 10 μm HNE or 1 mmFeSO4 (v), and PC12-Bcl2 cultures exposed to 10 μm HNE or 1 mm FeSO4(b) were separated by SDS-PAGE (100 μg protein/lane). Then proteins were transferred to a nitrocellulose sheet and immunoreacted with an antibody against HNE–protein conjugates. Note that HNE and FeSO4 induced the appearance of many HNE–protein conjugates in PC12-V cells, but not in the PC12-Bcl2 cells. D, PC12-V cells, PC12-Bcl2 cells, and primary hippocampal neurons were exposed for 2 hr to 0.2% ethanol (Control), FeSO4 (1 mm for PC12 cells and 2 μm for hippocampal neurons), Aβ (50 μm for PC12 cells and 10 μm for hippocampal neurons), or HNE (10 μm for PC12 cells and 2 μm for hippocampal neurons). Then cells were fixed and immunostained with HNE antibody, and relative levels of HNE immunoreactivity were quantified (see Materials and Methods). Values represent the mean and SEM of determinations made in four separate cultures per condition (100 cells scored/culture).