Impaired induction of HIF-1α in Psen1-/- immortalized mouse embryonic fibroblasts. In panel A, the time course of HIF-1α induction in Psen1+/+ (wt) and Psen1-/- (-/-) immortalized mouse embryonic fibroblasts is shown. Cultures were treated with 100 μm cobalt chloride for the indicated times (min). Lysates were prepared and Western blotting was performed using an anti-HIF-1α antibody. The lower panel shows the blot reprobed for β-tubulin. A representative blot is shown from experiments that were performed multiple times. In panels B and C, Psen1+/+ (wt) and Psen1-/- (-/-) cultures were treated with 100 μm cobalt chloride for 4 hours and analyzed for HIF-1α as in (A). The lowest panel in B shows representative samples of wt and -/- fibroblasts probed with the Psen1 antibody 33B10 to confirm the lack of detectible Psen1 expression in Psen1-/- fibroblasts. Panel C shows quantitation of the levels of HIF-1α in data derived from four independent experiments. In panels D and E cultures were serum starved overnight and then treated with insulin for 4 hours after which lysates were prepared and Western blotting performed as in (A). Panel E shows quantitation of the experiment shown in (D). In panel F, Psen1+/+ and Psen1-/- fibroblasts were treated with BAPTA-AM for 1.5 hours. Results from an experiment performed in triplicate is shown. The lower panel shows the blot reprobed for β-tubulin. All panels show representative blots from experiments that were performed multiple times. Western blots for HIF-1α were performed using a rabbit polyclonal antibody. The quantitative results are expressed as the ratio of HIF-1α to β-tubulin and are presented as + SEM. Asterisks in panels C and E indicate values that are different from the corresponding untreated cells (p < 0.05, unpaired t-tests). Other statistical comparisons are discussed in the text.