Fig. 4.

FOXO3a is phosphorylated and inhibited in BCR-ABL-expressing BaF3 cells. BaF3 (lanes 2–5) and BaF3P210 (lanes 6–8) cells starved from either IL-3 alone (in RPMI with 10% FCS; lanes 2, 3, 6, and 7) or IL-3 and serum (in RPMI with 1% BSA; lanes 4, 5, and 8) for 8 h were stimulated with 50 ng/ml IL-3 for 5 min (lanes 3, 5, and 7). Cell lysates were prepared as described (15), fractionated on a gradient SDS/PAGE, and transferred to nitrocellulose, and blots were probed with antiphospho-Thr-32, [1:750; anti-phospho-Ser-253, 1:500 (Upstate Biotechnology)] or antiphospho-Ser-315 (1:750; kindly provided by M. Greenberg) or with antitotal FOXO3a antibody. Bound antibodies were detected by the enhanced chemiluminescence system (DuPont–NEN). A control lysate of 293 T cells overexpressing FOXO3a WT was used as a positive control.