IGF-I induces Rho-GTP formation in H929 cells. H929 cells were starved in serum-free medium for 12 hours and were not stimulated or stimulated with varying concentrations of IGF-I (10 ng/mL to 200 ng/ml; A) or with 100 ng/mL IGF-1 for different times (5 min to 120 min; B). Fresh lysates were then affinity precipitated (AP) using GST-RBD glutathione beads or GST-PBD glutathione beads. After washing 3 times, the bound proteins were eluted and analyzed by Western blotting using RhoA-, Rac-, and cdc42-specific antibodies. The same lysates were directly subjected to SDS-PAGE, transferred to membranes, and blotted with anti-Rho antibodies as a control for loading.