FIGURE 7.

SFKs and SHP-2 are required for IGF-IR internalization. Serum-starved MCF-7 cells were pretreated with dimethyl sulfoxide (DMSO) and either 10 μm SU6656 (A) or 50 μm NSC-87877 (B) or were transfected with nontargeting (NT) or SHP-2-specific siRNA duplexes (C) and labeled with 0.25 mg/ml sulfo-NHS-biotin at 4 °C for 2 h. The cells were then treated with IGF-I at 37 °C for 5 min to allow for internalization of IGF-IR. Plasma membrane-associated biotin was then cleaved with dithiothreitol (DTT). Biotinylated proteins that had internalized were immunoprecipitated using NeutrAvidin-conjugated beads and subjected to Western analysis. Top panels, representative experiment; bottom panels, quantitated internalized IGF-IRβ. The asterisks denote significant differences in internalized biotinylated IGF-IRβ compared with IGF-I treatment. *, p < 0.05; **, p < 0.01 (two-way ANOVA, Bonferroni post-test).