Bach2 is phosphorylated via the PI-3K/S6K pathway in vivo. (A) Lysates of Ramos cells were exposed (+) or not (-) to 20 units of alkaline phosphatase (AP) for 30 minutes. (B) BV173 and Ramos cells were exposed (+) or not (-) to 1 μM imatinib (IM) for 24 hours. (C) Ramos cells were either treated (+) or not (-) with 25 μM LY294002 (LY) or DMSO for the indicated time. BV173 cells were either treated (+) or not (-) with 50 μM LY or DMSO for 24 hours. (D) 3T3 cells were transiently transfected with pcDNA3.1Bach2. After serum starvation for 6 hours, the cells were either treated (+) or not (-) with 50 μM LY or 80 nM rapamycin (Rap) for 30 minutes and then stimulated (+) or not (-) with serum for 30 minutes. (E-F) 3T3 cells were transiently transfected with pcDNA3.1haBach2 together with pRK7, pRK7/HAS6K1E389D3E, pcDNA3.1, or pcDNA3.1S6K2T401D. After serum starvation for 6 hours, the cells were either treated (+) or not (-) with 50 μM LY for 30 minutes and then stimulated (+) or not (-) with serum for 30 minutes. (A-F) Total cell lysates were separated on SDS-PAGE and immunoblotted with a Bach2 monoclonal antibody, phospho-Akt (S473) (P-Akt), Akt, phosho-p70S6K (T389), or p70S6K antibodies. Phosphorylated (P-Bach2) and unphosphorylated Bach2 are indicated by the arrows. In panel E, the Bach2 strip for lanes 1 to 4 was taken from a 20-minute autoradiograph exposure and, for lanes 5 to 8, from a 5-minute exposure to compensate for different strengths of the Bach2 signal resulting from different efficiencies of cotransfection with the empty vector (pRK7) or the S6-K1 construct (pRK7/HAS6K1E389D3E). All images are representative of at least 3 independent experiments.