Fig 1: IGFBP2 increases IGF1-dependent phosphorylation of AKT in a dose-dependent manner. Monolayers of HEK293-2 cells (A) or HuH-7 cells (B) were preincubated for 24 h with different concentrations of IGFBP2 (0, 33.75, 67.5, 125, 250, 500, 1000, and 2000 ng/mL) in culture medium with 0.5% serum. For the bioassay, cell monolayers were incubated with 10 ng/mL IGF1 (blue) or 100 ng/mL IGF1 (red) for 20 min then lysed and assayed for phosphorylated AKT. Significance is provided for the comparison of different IGF1 or IGFBP2 concentrations (n = 3, means ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). In one case, one outlier was identified by GraphPad Prism and not used for the calculation of the mean (n = 2 for HuH-7 cells preincubated in 2000 ng/mL IGFBP2 and challenged by 100 ng/mL IGF1; for abbreviations please also see Figure 1).
Fig 2: Control of IGF-related bioactivity by inhibitors (STC1 and STC2), proteases (PAPPA and PAPPA2), and IGFBPs (IGFBP1 to 6). IGF-dependent activation of the IGF1 receptor is quantified by the KIRA assay. IGF-related activation of AKT can be assessed by the novel BIRA assay (STC1 and 2: stanniocalcin 1 and 2; PAPPA and A2: pregnancy-associated protein protease A and A2; IGF: insulin-like growth factor; IGFBP: IGF-binding protein; MAPK: mitogen-activated protein kinase; AKT: protein kinase B; KIRA: kinase receptor activation; BIRA: IGFBP2-enhanced IGF-related AKT activation).
Fig 3: Dose-dependent increase of AKT phosphorylation in IGFBP2-transfected HEK293-10 cells. Cells were incubated in different concentrations of human recombinant IGF1 (0, 10, 100, 300 ng/mL) for 20 min before they were lysed and assayed for phosphorylation of AKT by Western immunoblotting, as described in Materials and Methods (mean ± SEM; n = 23, * p < 0.05, ** p < 0.01, **** p < 0.0001; abbreviations are explained in Figure 1).
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