Constitutive Igbp1 expression increases phosphorylation of 4EBP and S6K and enhances Epo-induced polysome recruitment of structured transcripts. (A,B) I/11 cells transduced with an empty control vector (vector) or with an Igbp1 expression vector were factor-deprived (4 hours), stimulated with Epo (E, 5 U/mL), SCF (S, 100 ng/mL), or Epo plus SCF (ES), or left untreated (NF). Expanding I/11 cells in the presence of Epo, SCF, and dexamethasone are steady state (ss). (A) Western blots from total cell lysates were stained with antibodies recognizing total 4EBP (4EBP Ab). The nonphosphorylated, hypophosphorylated, and hyperphosphorylated proteins can be discriminated by their distinct electrophoretic mobility as α, β, and γ isoforms, respectively. (B) Western blots from samples stimulated as indicated for 10 minutes were stained with a phospho-specific antibody against p70S6K (P-S6K) and counterstained for total S6K to control for equal loading. (C-H) Expanding I/11 empty vector control cells (■) or cells constitutively expressing Igbp1 (□) were factor-deprived and left untreated (NF) or restimulated with erythropoietin (Epo, 2 U/mL; 2 hours). Free and polysome-bound mRNA was isolated and assayed for the expression of Fli-1 (C), Igbp1 (D), eEF1β (E), rps4 (F), Nm23 (G), and mEd2 (H). The percentage of mRNA associated with polysomes (pb-mRNA) was calculated for the different genes under the different conditions. Constitutive Igbp1 expression enhances polysome recruitment of translationally controlled transcripts in response to Epo alone.