Expression and activation status of Bcl-2 family members in NPM/ALK-expressing cells. (A) Expression and tyrosine phosphorylation of NPM-ALK were evaluated with an anti-ALK monoclonal and antiphosphotyrosine (PY) antibodies in total lysates from pro-B Ba/F3 parental cells cultured in the absence or presence of IL-3 for 18 hours (lanes a-b), in NPM/ALK-expressing cell lines including Ba/F3 cells transfected with NPM/ALK (Ba/F3-N/A; lane c), and the human ALCL-derived cell lines SUP-M2 (lane d), K-299 (lane e), and SUDHL-1 (lane f). Expression levels of Bcl-X_L, Bcl-2, and Bad were also assessed. (B) Ba/F3 parental cells were starved without IL-3 for 2 hours (lane a) and then stimulated with IL-3 in the absence (lane b) or presence (lane c) of 25 μM 294002 for 30 minutes. Ba/F3-N/A⁺ and K-299 ALCL cells were incubated with the same drug concentration (lanes e, g) or vehicle alone (lanes d, f) for 30 minutes. Expression and activation status of AKT and Bad were evaluated from total lysates with anti-total AKT and Bad polyclonal antibodies as well as by using antiphosphoserine-specific antibodies (phospho-Ser473 for AKT and phospho-Ser136 for Bad).