OLFM4 inhibits 4E-BP1 phosphorylation. (A) HL-60 cells were transfected with OLFM4 expression plasmid (OLFM4) or empty vector (vec) and selected by G418. G418-resistant HL-60 cells were treated with ATRA (1μM) for 3 days. Total cell lysates were subjected to Western blotting analysis with antibodies for phospho-p44/42 MAPK (Thr202/Tyr204), phospho-Akt (Ser473), phospho-GSK3β (Ser9), phopho-p70S6 kinase (Thr389), and phospho-4E-BP1 (Ser65 or Thr70). The blots were stripped and reprobed with corresponding antibodies for p44/42 MAPK, Akt, GSK-3β, p70S6 kinase, and 4E-BP1. (B) HEK-293T cells were cotransfected with 4E-BP1 or p70S6 kinase expression plasmid together with OLFM4 expression plasmid in different amounts, as indicated. After 24 hours, total cell lysates were subjected to Western blotting analysis with antibodies for phospho-4E-BP1 (Ser65, Thr70, or Thr37/46) or phospho-p70S6 kinase (Thr389). The blots were stripped and reprobed with total anti–4E-BP1 or anti-p70S6 kinase. (C) AML-193 cells were transduced with lentiviral shRNA against OLFM4 or control shRNA. After 48 hours, total cell lysates were subjected to Western blotting analysis with antibodies for OLFM4, phospho-4E-BP1 (Ser65, Thr70, or Thr37/46), phospho-Akt (Ser473), or phospho-p70S6 kinase (Thr389). The blots were stripped and reprobed with corresponding 4E-BP1, Akt, and p70S6 kinase antibodies.