HA-BAD in parental 32D cells.

(A) Phosphorylation of HA-BAD in parental 32D cells treated with phosphatase inhibitors. 32D cells were pretreated either with cyclosporin A (CyA) (0.5 μmol/L), or cantharidin (Cant) (high [cant-high], 30 μmol/L; low[cant-low], 250 nmol/L) or calyculin A (20 nmol/L) for 2 hours and then deprived from IL-3 for 4 hours. At 0 and 4 hours, cells were harvested and lysed. Phosphorylated BAD was detected by using a mix of antiphospho BAD antibodies. Levels of HA-BAD were measured as control of loading. (B) Association between HA-BAD and 14-3-3β. 32D cells were pretreated with phosphatase inhibitors (see above) and then starved from IL-3. The HA-BAD: 14-3-3β interaction was evaluated in cytoplasmic extracts 0 and 4 hours after IL-3 withdrawal. Levels of immunoprecipitated HA-BAD were measured as control of loading. (C) BAD expression levels in subcellular fractions of parental cells treated with phosphatase inhibitors. Western blots show exogenous BAD levels in fractions enriched for cytoplasm (C) and mitochondria (HM) from untreated and phosphatase inhibitor-treated 32D cells at 0 and 4 hours after IL-3 withdrawal. Levels of mitochondrial marker subunit IV of cytochrome oxidase (COX) and cytoplasmic marker HSP90 were measured as control of equal loading and purity of subcellular fractions. Representative of 3 different experiments.