Fig. 4

Activation of IGF-1R is abrogated after in vitro treatment with IGFBP6. a TMZ-sensitive (U251) and TMZ-resistant (UTMZ) cells were cultured in serum-free medium (SFM) for 16 h, then stimulated by IGF2 (50 ng/ml) for the indicated time. Cell lysates were subjected to Western blot analysis to determine the phosphorylation of IGF-1R and AKT (top panel) and total IGF-1R and AKT (bottom panel) in TMZ-resistant cells. AKT was constitutively activated in TMZ-sensitive cells. Representative blots are shown. b Quantitative analysis of protein expression levels from blots shown in (a). Bars represent the average from triplicate determinations from at least three independent experiments. c TMZ-resistant cells were starved in SFM overnight and then stimulated with IGF2 (50 ng/ml) in the presence or absence of recombinant IGFBP6 (100 or 200 ng/ml). Representative Western blots showing that IGFBP6 (but not IGFBP2) abrogated the IGF2-dependent phosphorylation of IGF-1R and AKT. Expression of Rab11 is shown as the loading control