IGF1 induced IGF1R phosphorylation and downstream signaling.

<p>A, Wild type and L1361R mutant IGF1R autophosphorylation in transiently transfected R⁻ cells after stimulation with 10 nM IGF1 for 0–30 minutes. Immunoblots were incubated with specific antibodies against phosphorylated IGF1R β-subunit (P-IGF1R), Akt (P-Akt) or Mapk/Erk (P-Mapk/Erk), stripped and subsequently incubated with specific antibodies against the IGF1R α-subunit (IGF1R), Akt (Akt), or Mapk/Erk (Mapk/Erk). IGF1R α-subunit levels were assessed as control of successful transfection. Densitometric units were normalized to total levels of IGF1R α-subunit and the fold increase was calculated (IGF1R-WT at 0 min was set 1). Results are shown as means ± SEM calculated from four independent experiments. B, Yeast two-hybrid analysis of the interaction of the IGF1R derivatives (IGF1R-C-WT, IGF1R-C-L1361R, IGF1R-C-KD) with adapter proteins (IRS1, 14-3-3ß, GIPC, PI3Kp85) by lift-off assay (left panel). Positive interactions led to blue staining (here black) of the colonies. POS, positive control p53 x large T-antigen; NEG, negative control Lamin x large T-antigen. The filter assays shown are representative for more than three independent experiments. Quantification of protein-protein interaction was measured using ONPG-assays and data are shown as mean ± SEM (right panel).