FIGURE 2.

Rapamycin inhibits IGF-1-stimulated cellular protein expression of RhoA, Cdc42, and Rac1 by suppressing their protein synthesis. A, Rh30 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 semiquantitative RT-PCR. PCR products were separated on 1% agarose gel and stained with ethidium bromide. Bands were visualized under UV light and photographed with a digital camera. The effects of rapamycin on protein synthesis (B) and degradation (E) in Rh30 cells were determined as described under “Experimental Procedures.” Semiquantitative data for A, B and E by densitometry using NIH ImageJ are shown in C, D and F, respectively. 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). CHX, cycloheximide.