Figure 3

SNS-032 strongly inhibits expression and phosphorylation of downstream targets of mTOR, and removal of SNS-032 correlates with the recovery from inhibition of phosphor-mTOR and cell death. (A) Exponentially growing cells exposed to the indicated doses SNS-032 for 6 h and then cell lysates were prepared, and 50 μg of total cell extracts was separated in 6-12% SDS-PAGE. Protein bands were detected with antibodies to phosphor-Akt (Ser437/Thr308), Akt, phosphor-4E-BP1 (Ser65/Thr37/46/Thr70), 4E-BP1, phosphor-p70S6K (Thr389), p70S6K, and actin. This experiment was repeated once with identical results. (B) KG-1 cells were treated with 100 nM SNS-032 for 6 h, and then divided into two parts. One was washed into fresh medium without the drug, whereas the other was continuing to culture without washing. The cells were collected at the indicated times for Western blot analysis.