Polysome recruitment of Igbp1 and mEd2 is PI3K and eIF4E sensitive. I/11 cells were factor-deprived for 4 hours and subsequently restimulated with Epo (E, 2U/mL), SCF (S, 100 ng/mL), or both (ES) for 2 hours. Where indicated, LY294002 (LY; 10 μM) or rapamycin (R; 10 nM) was added 30 minutes before and during restimulation. Total RNA as well as free and pb RNA fractions were isolated, and gene expression was measured by real-time PCR. (A-C) The expression ratio in restimulated versus factor-deprived cells is calculated as a log-2 fold-change for Igbp1 (A), mEd2 (B), and Fli1 (C); in total (□) and pb mRNA (■). (D-F) The percentage of mRNA associated with polysomes (pb-mRNA) was calculated for the same genes under the different conditions. (G-I) I/11 cells transduced with an empty control vector (■) or with an eIF4E expression vector (eIF4E overexpression, □) were factor-deprived and restimulated as indicated. The percentage pb mRNA was calculated for Igbp1 (G), mEd2 (H), and Fli1 (I). (J-L) I/11 cells were induced to differentiate and total RNA was isolated before (t0) and 24 (t24), 48 (t48), and 72 hours (t72) after differentiation induction. The expression ratio in differentiated vs nondifferentiated cells was calculated as a log-2 fold-change for Igbp1 (J), mEd2 (K), and Nfe2 (L). Error bars were calculated from 4 independent measurements using 2 different RNA batches.