HEK293 Tet-On HA-REDD1 cells (A) and HEK293 Tet-On control cells (B) were treated with doxycycline (Dox) for the time periods indicated. (C) HEK293 Tet-On HA-REDD1 cells were treated with Dox for 6 h followed by cycloheximide for up to 30 min. Abundance of HA-tagged and total REDD1, p70S6K1, 4E-BP1, GAPH, phosphorylation of p70S6K1 on Thr389 and 4E-BP1 on Ser65 were assessed in whole cell lysates by Western blot analysis. Blots shown in panels A-C are representative of results for two experiments (N=2); within an experiment, two independent samples were analyzed.