Analysis of the mutual activation of Ras, PI 3-K, and Akt in the parental Ba/F3 cells and the transfectants.

(A) Parental Ba/F3 cells and other transfectants were cultured in the media without IL-3 overnight. Cell extracts prepared from the indicated cell lines were analyzed by Western blotting using antiphosphoserine-Akt antibody. The membrane was reprobed with pan-Akt antibody. (B) Parental Ba/F3 cells and other transfectants were cultured in the media without IL-3 for 4 hours. Cell extracts prepared from the indicated cell lines were subjected to pull-down analysis using GST-Raf fusion protein. Precipitates were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted with anti-Ras antibody. (C) Parental Ba/F3 cells and transfectants expressing p110CAAX alone or p110CAAX/v-Abl Δ858-1080 were subjected to PIP₃ assay as described in “Materials and methods.”