Figure 3.

Temporal profiling of nuclear P-AKT, HIF-1α, and P-CREB. (A) IGF-1 stimulated the nuclear translocation of P-AKT after 2, 4, 6, and 24 hours in the 184htert cells. The controls for this experiment were SK-N-MC cells treated with forskolin (positive) and without forskolin (negative). Neither of the controls received IGF-1. (B) Temporal profiling of HIF-1α in the absence or presence of IGF-1. IGF-1 stimulated the translocation of HIF-1α at the 4- and 6-hour time points. (C) Temporal profiling of P-CREB. IGF-1 was added to the 184htert cells for 0, 1, 3, and 6 hours and the protein extracts were evaluated for P-CREB (top panel) and total CREB (bottom panel). CREB was maximally phosphorylated after 1 hour following IGF-1 treatment while there was no effect on ATF-1. The total amount of CREB protein was the same between time points (bottom panel). (D) Inhibition of AKT signal transduction with LY294002 (30 µM) inhibited the phosphorylation of CREB by IGF-1. Cells were pretreated with LY294002 for 10 minutes then IGF-1 was added for 1 hour. Desferrioxamine (DXF, 100 µM) was added to the cells for 1 hour as a positive control for p-CREB. An equal volume of dimethyl sulfoxide (DMSO) served as a vehicle control for the LY294002 compound. There was no inhibition of P-CREB in the presence of DMSO and IGF-1.