Effect of rapamycin on the phosphorylation of mTOR, p70S6K, and 4E-BP1. CD34⁺ cells were cultured in the presence of TPO without Rapa (TPO) or with Rapa (100 nM) added to the culture at day 0 and day 3 (TPO + Rapa). Cells were taken at day 6 to perform Western blot analyses. (A) Effect of Rapa on Ser2448 phosphorylation of mTOR. (B) Effect of Rapa on Thr389 of p70S6K. Inhibition of Thr389-phosphorylation by Rapa was reversible as shown after removing the medium containing TPO and Rapa at day 6, washing the cells twice in PBS, and restimulation for 1 day with fresh medium supplemented with TPO alone (TPO + Rapa/TPO). (C) Effect of Rapa on Thr37/46 of 4E-BP1. Accumulation of unphosphorylated (α), phosphorylated (β), and diminution of hyperphosphorylated (γ) forms is shown.