Figure 6.
Preloading of cells with ascorbate hinders HIF-1α protein stabilization by Ni(II) exposure. A. 1HAEo- cells, or B, A549 cells were preloaded with 25 μM ascorbate for 2 h, washed to remove extracellular ascorbate and exposed to 0.5 mM NiSO4 for various time periods shown in the Figure. Right panel HIF-1α and HIF-1β induction in ascorbate preloaded cells. Left panel cells without ascorbate preloading shown for comparison. Nuclear protein extracts (15 μg) were prepared for immunoblotting as described in Materials and Methods and probed with antibodies against HIF-1α, HIF-1β. Antibodies against β-actin were used to provide loading control.