Figure 2.

Transactivation of the IRE by IRE-BP1 and by insulin. A, SK-HEP-1 cells were transfected with pGL2 basic vector alone, wild-type (WT) IGFBP-3 −1200/+34-luciferase reporter or with sequences containing mutations of IGFBP-3 IRE (mutants 1–6, substituted sequences shown in supplemental Table 1) in the absence (white bars) or presence (black bars) of IRE-BP1 expression vector, followed by luciferase reporter assay. Relative luciferase activity was corrected for protein concentration in the lysate. n = 3 each group. This experiment was conducted three times with similar results. B, WT IGFBP-3 −1200/+34 and mutants 1–6 were transfected into SK-HEP-1 cells without IRE-BP1 expression vector. Cells were then treated with vehicle (white bars) or with 10 nm insulin (black bars) overnight and luciferase activity measured as above. n = 3 replicates per condition. The experiment was conducted four times with similar results.