GSK690693 inhibits AKT signaling in ALL cells. (A) I2.1, I9.2, A3, and Tanoue cells were treated with DMSO or different concentrations of GSK690693 for 6 hours. Cell lysates were analyzed by Western blot analysis for phospho-GSK3β Ser9, phospho-PRAS40 Thr246, phospho-p70S6K Thr421/Ser424, phospho-AKT Ser473, and phospho-AKT Thr308. Total AKT and tubulin are shown as loading controls. (B) GSK690693 inhibits AKT kinase activity in ALL cells. A3 and Tanoue cell lines were treated with DMSO or various concentrations of GSK690693 for 1 hour. AKT was immunoprecipitated from cell lysates with immobilized anti-AKT antibody, and the kinase assay was performed using GSK-3 fusion protein as a substrate. Phosphorylation of GSK3 was measured by Western blot analysis, using phospho-GSK3β antibody. (C) GSK690693 inhibits stimulated AKT kinase activity in ALL cells. Tanoue cells were serum-starved overnight, then treated with DMSO or 1 μM GSK690693 for 1 hour. Cells were treated with 5 ng/mL insulin for various times to stimulate AKT activity. AKT kinase assay was performed.