Fig. 5.
Abrogation of IGF2-mediated phosphorylation of IGF-1R is mediated by IGFBP6 binding and sequestration of IGF2. a Schematic representation of human IGFBP6 (Swiss-Prot, P24592) showing mutated sites. Amino acids mutated to Ala are shown in red. b Western blot analysis of recombinant IGFBP6 proteins (top panel). wt, wt-IGFBP6; mut, mut-IGFBP6; wtc, commercial wt-IGFBP6. Western blot ligand binding analysis (bottom panel). Wt-IGFBP6 (250 ng) and mut-IGFBP6 (250 ng or 1 μg) proteins were separated on 4–20% gels and transferred to PVDF membranes, incubated with Strep-Tag II-IGF2 and then developed with NWSHPQFEK Tag antibody. c Representative Western blot showing that wt-IGFBP6 (but not mut-IGFBP6) abrogates the IGF2-dependent phosphorylation of IGF-1R and AKT in TMZ-resistant cells. UTMZ cells were cultured in SFM for 16 h and then stimulated with IGF2 (50 ng/ml) in the presence or absence of recombinant wt-IGFBP6 or mut-IGFBP6 (200 ng/ml). d Representative Western blot showing that conditioned medium from chemosensitive cells abrogates the IGF2-dependent phosphorylation of IGF-1R and AKT in chemoresistant cells. UTMZ cells were cultured in SFM overnight and then stimulated with IGF2 at the indicated doses in the presence of conditioned medium from U251 cells. Expression of Rab11 is shown as the loading control. e Quantitative analysis (top) of extracellular IGFBP6 in conditioned medium of UTMZ cells stably transfected with one of five IGFBP6-shRNA constructs or a non-targeted control shRNA construct. Extracellular IGFBP6 was detected by Western blot (bottom) after 48 h of culture in SFM. Data are presented as the mean ± SEM of three independent experiments (*P < 0.05, **P < 0.001). f Representative Western blot showing that abrogation of the IGF2-dependent phosphorylation events requires the binding and sequestration of IGF2 by IGFBP6. UTMZ cells were cultured in SFM overnight and then stimulated with IGF2 at the indicated doses in the presence of conditioned medium from TMZ-sensitive U251 cells transfected with control shRNA or IGFBP6-shRNA constructs. Expression of Rab11 is shown as the loading control. CM, conditioned medium; CS, TMZ-sensitive cells; NT, non-targeted