Functional characterization of ATIC-ALK.

(A) An antiphosphotyrosine antibody (αPTyr) immunoblot of transfected COS7 cell lysates immunoprecipitated using either preimmune (PI) serum or anti-ALK #11 (αALK) rabbit polyclonal antiserum demonstrates constitutive tyrosine phosphorylation of the approximately 87-kd ATIC-ALK and 80-kd NPM-ALK chimeric proteins. (B) Immunofluorescent microscopy of COS7 cells transiently transfected with pcDNA3-ATIC-ALK, pcDNA3-NPM-ALK, or empty vector and stained with the ALK1 monoclonal antibody. Subcellular localization of the expressed proteins, as indicated by green fluorescent signal from the FITC (fluorescein isothiocyanate)-labeled secondary antibody, shows ATIC-ALK to be present in the cytoplasm only, whereas NPM-ALK is found in both the cytoplasm and nucleus. Propidium iodide (PI) staining of DNA was performed simultaneously to identify nuclei. (C) ATIC-ALK confers factor-independent growth to BaF3 cells. Stably transfected BaF3 pools expressing ATIC-ALK or NPM-ALK were assessed for growth in the presence or absence of IL-3, together with empty vector-containing cell pools. Viable cell counts were performed in triplicate using trypan blue at 24-hour intervals, with each point being the average of the triplicate determinations.