RA induces phosphorylation of p70 S6 kinase in NB-4 cells and primary APL blasts, but not in the RA-resistant NB-4.007/6 (NB-4R) cell line. (A) NB-4 or NB-4.007/6 cells were treated with RA (1 μM) for the indicated times. Equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated/activated form of p70 S6 kinase on threonine 421 and serine 424. (B) The blot shown in panel A was stripped and reprobed with an anti-p70 S6 kinase antibody, to control for protein loading. (C) RA activates the kinase domain of p70 S6 kinase in NB-4, but not NB-4R, cells. NB-4 and NB-4R cells were treated with RA for 48 hours. The cells were subsequently lysed, and equal amounts of protein were 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. Kinase activity is expressed as CPM (counts per minute) after normalizing for nonspecific activity present in RIgG immunoprecipitates. Means plus or minus the standard error (SE) of 2 independent experiments are shown. (D) Isolated peripheral blood mononuclear cells from a patient with acute promyelocytic leukemia were treated with RA (1 μM) for the indicated times. Equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated/activated form of p70 S6 kinase on threonine 421 and serine 424. (E) The blot shown in panel D was stripped and reprobed with an anti-tubulin antibody, to control for protein loading.