FIGURE 1.

Rapamycin inhibits IGF-1-stimulated protein expression and activities of RhoA, Cdc42, and Rac1. Indicated cells were serum-starved for 24 h and then pretreated with or without rapamycin (100 ng/ml) for 2 h followed by stimulation with or without IGF-1 (10 ng/ml) for 22 h. Cells were harvested for the GTPase activity assay (A) and Western blot analysis (B) for RhoA, Cdc42, and Rac1 as described under “Experimental Procedures.” Densitometry for the bands in A and B was performed using NIH ImageJ, as shown in C and D, respectively. Note: for calculation of the ratio of GTP-bound/total protein of the small GTPases, shown in C, total protein values used were from D. Results are the means ± S.E. and are pooled from three independent experiments. ^a, p < 0.05, difference versus control group; ^b, p < 0.05, difference versus IGF-1 group (unpaired Student's t test).