RA-dependent phosphorylation/activation of the Akt-kinase and phosphorylation of the 4E-BP1 repressor of mRNA translation. (A) NB-4 cells were incubated with RA for the indicated times. Two and a half hours prior to cell lysis, LY294002 was added to the cultures, as indicated. The cells were subsequently lysed and equal amounts of total cell lysates were resolved by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of Akt on serine 473. (B) The blot shown in panel A was stripped and reprobed with an anti-Akt antibody, to control for protein loading. (C) NB-4 or NB-4 0.007/6 (NB-4R) cells were incubated with RA for the indicated times. The cells were lysed and equal amounts of total cell lysates were resolved by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of Akt on serine 473. (D) The blot shown in panel C was stripped and reprobed with an anti-Akt antibody, to control for protein loading. (E-L) NB-4 cells were incubated with RA for the indicated times. Two and a half hours before lysis, the indicated inhibitors were added in the culture. The cells were subsequently lysed, and equal amounts of cell lysates were analyzed by SDS-PAGE and immunoblotted with an anti-phospho-Thr37/46-4E-BP1 antibody (E) or an anti-phospho-Ser65-4E-BP1 antibody (G, K), or an anti-phospho-Thr70-4E-BP1 antibody (I). The same blots were subsequently stripped and reprobed with an anti-4E-BP1 antibody (F, H, J, and L, respectively).