Bach2 is phosphorylated on S521 by the PI-3K/S6K pathway. (A) 3T3 cells were transfected with expression vectors expressing wild-type or mutants of Bach2. Total cell lysates were separated on 5% SDS-PAGE and immunoblotted with a Bach2 monoclonal antibody. (B) Comparison between the consensus aa sequence for S6K phosphorylation and Bach2 aa's 514 to 522. (C) 3T3 cells were transiently transfected with pcDNA3.1HABach2 (lanes 1 to 4) or pcDNA3.1HABach2S521A (lanes 5 to 8) and (D) were also cotransfected with pRK7/HAS6K1E389D3E. After serum starvation for 6 hours, the cells were either treated (+) or not (-) with 50 μM LY294002 (LY) for 30 minutes and then either stimulated (+) or not (-) with serum for 30 minutes. Total cell lysates were separated on SDS-PAGE and immunoblotted with a Bach2 monoclonal antibody, antiphospho-Akt (S473), or Akt antibodies. Phosphorylated (P-Bach2) and unphosphorylated Bach2 are indicated by the arrows. All images are representative of at least 3 independent experiments.