Tyrosine kinase activity of NPM/ALK up-regulates Bcl-XL and promotes Bad phosphorylation. Ba/F3-NPM/ALK+ cells were transiently transfected with NPM/ALK kinase defective mutant (N/A.K210R), an empty control vector (N/A.EV), or electroporated in the absence of DNA (Ba/F3.N/A). (A) At 48 hours from transfection total lysates were separated by 7.5% SDS-PAGE and analyzed by immunoblotting with anti-ALK (ALK) and antiphosphotyrosine (PY) monoclonal antibodies (upper panel). The same cellular lysates were also separated by 12% SDS-PAGE and analyzed by immunoblotting with antibodies specifically recognizing total Bcl-XL, Bcl-2, Bad, and a phosphoserine-136 (p136-Bad) residue of Bad (lower panel). Levels of β-actin were reported as a protein loading control (lower panel). (B) Cell-cycle distribution of the transfected cells was assessed 48 hours after transfection by propidium iodide staining and flow cytometric analysis. The sub-G1 peak is indicative of the presence of apoptotic cells with subdiploid DNA content. (C) Occurrence of apoptosis was also performed by a propidium (Prop.) iodide-annexin V staining at 48 hours after transfection.
