FIG. 8.

Prolonged rapamycin treatment only mildly represses the translation of TOP mRNAs. (a and d) Proliferating 293 cells were treated with 20 nM rapamycin for the indicated time (a) or for 1 h (lane R in d) or with 50 mM LY294002 for 30 min (lane L in d), after which the cells were harvested. The cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies. The bottom panel in d is the Ponceau S-stained membrane, showing the relative protein loading among samples. (b and c) Proliferating 293 cells were treated with 20 nM rapamycin for the indicated times (b) or for 4 h (c) or with 50 mM LY294002 for 30 min. The polysomal distribution of rpL32, eEF1A, and actin mRNAs was analyzed as described in the legend to Fig. 1. The relative amount of the mRNAs in polysomes is presented graphically in b and numerically in c.