Altered induction of HIF-1α in Psen1-/- primary neuronal cultures. Primary neuronal cultures were prepared from E15.5-16 embryonic brains and maintained in Neurobasal medium/B27 supplement. Experiments were performed after 5-6 days in vitro. In panel A, cultures were treated with 100 μm cobalt chloride for the indicated times (hrs). In panels B and C, cells were cultured without B27 overnight and then treated with insulin (B) or IGF-1 (C) for six hours. In panel D, cells were cultured without B27 overnight and then treated with IGF-1 for the indicated times. Western blotting for HIF-1α was performed as in Figure 1 and the lower panels show the same blots reprobed for β-tubulin or actin. In panels E and H, the time course of the studies in (A) and (D) are quantitated. The insulin and IGF-1 studies in panels B and C are quantitated in (F) and (G). All panels show representative blots from experiments that were performed multiple times. Asterisks indicate p < 0.05 vs. untreated control (unpaired t-tests).