Activation of the kinase domain of the p70 S6 kinase by RA is FRAP/mTOR- and PI3′K-dependent, and PI3′K activity is required for differentiation of APL cells. (A) NB-4 cells were treated with RA for 48 hours. Two and a half hours prior to cell lysis, the indicated inhibitors were added in the cultures. The cells were subsequently lysed, and immunoprecipitated with an anti-p70 S6 kinase antibody or nonimmune rabbit immunoglobulin (RIgG). In vitro kinase assays to detect p70 S6K activity were subsequently carried out on the immunoprecipitates. The data are expressed as fold increase over untreated controls, and represent the mean plus or minus SE of 2 independent experiments. (B) NB-4 cells were incubated with RA for the indicated times. Total cell lysates were resolved by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of mTOR on serine 2448. (C) The blot shown in panel B was subsequently stripped and reprobed with an anti-mTOR antibody, to control for protein loading (right panel). (D) NB-4 cells were treated with RA for the indicated times. Two and half hours prior to cell lysis, rapamycin (20 nM) or LY294002 (50 μM) were added in the cultures, as indicated. Equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated/activated form of the p70 S6 kinase on threonine 421 and serine 424. (E) The blot shown in panel D was stripped and reprobed with an anti-p70 S6 kinase antibody, to control for protein loading. (F) NB-4 cells were incubated with RA, in the presence or absence of the PI3′K inhibitor LY294002. The cells were subsequently stained with an anti-CD11b monoclonal antibody and analyzed by flow cytometry. The data represent mean ± SE of 3 experiments.