HA-BAD in parental and BCR/ABL-expressing cells.

(A) Phosphorylation of HA-BAD in parental and BCR/ABL-expressing cells starved of both IL-3 and serum in [³²P]-orthophosphate-containing medium. Immunoprecipitations were carried out using an anti-HA antibody. Vector-infected BCR/ABL-expressing cells were used as negative control. (B) Association of 14-3-3β and HA-BAD in infected parental and BCR/ABL-expressing cells. Infected parental cells were starved of IL-3 and the interaction 14-3-3/BAD was assessed in cytoplasmic extracts 0, 30, and 60 minutes after the removal of IL-3. BCR/ABL-expressing cells infected with WT and DM BAD were starved for 4 hours and then lysed. Total extracts were analyzed for BAD-14-3-3β association. Lane C shows expression of 14-3-3β and HA-BAD in total extracts from BCR/ABL/WT BAD-expressing cells. (C) Levels of phosphorylated BAD after IL-3 removal. Cells were starved of IL-3 for 4 hours, harvested, and lysed at different times (0, 1, 2, 3, and 4 hours). Phosphorylated BAD was detected using a mix of antiphospho BAD antibodies. Levels of HA-BAD were measured as control of equal loading. (D) Stability of WTHA-BAD in starved 32D cells. Levels of [³⁵S]-labeled HA-BAD were assessed at 0, 1, 2, and 4 hours after the simultaneous removal of [³⁵S]-methionine and IL-3. Levels of HA-BAD were measured as control of immunoprecipitation efficiency. The asterisk (in B and D) indicates immunoglobulin light chain. Results are representative of 3 independent experiments.