γ-secretase activity is not needed for insulin or cobalt induction of HIF-1α. Psen1+/+ immortalized fibroblasts (A) were treated overnight with the γ-secretase inhibitor XXI and then stimulated with insulin for 6 hours. Western blotting for HIF-1α was performed (upper panel) followed by reprobing of the blot for β-tubulin (middle panel). The same samples were then reblotted and probed for N-cadherin (lower panel). A band for the full length N-cadherin (FL) is indicated. A band that corresponds to the N-cadherin CTF is visible in the γ-secretase inhibitor treated lanes. In panel B, Psen1+/+ fibroblasts were treated overnight with the γ-secretase inhibitor L-685,458 and then stimulated with cobalt chloride for 6 hours. The samples were blotted and probed for HIF-1α (upper panel) and β-actin (middle panel) and then reblotted and probed for N-cadherin (lower panel). In panel C, the experiment in (A) was quantitated with the levels of HIF-1α normalized to β-tubulin. Analysis of the data in panel C (n = 3 per group) revealed significant increases in HIF-1α following treatment with insulin vs. untreated control (p = 0.004, unpaired t-test) or XXI treated vs. insulin + XXI (p = 0.01). There was no difference between insulin treated vs. insulin plus XXI treated cultures (p = 0.29) although XXI treatment alone modestly decreased HIF-1α levels vs. untreated control (p = 0.006). Representative experiments are shown. Asterisks (*) indicate values that are significantly different from corresponding samples that were not treated with insulin.