Stat5 DNA binding is constitutively activated in TEL-Jak2 transformed Ba/F3 cells. Nuclear extracts were prepared from Ba/F3 (lanes 1 through 5), Ba/F3 HA-TEL-Jak2 JH1 subclone 36 (lanes 6 through 10), Ba/F3 HA-TEL-Jak2 JH2 subclone 4 (lanes 11 and 12), Ba/F3 HA-TEL-Jak2 JH2+JH1 subclone 23 (lanes 13 through 17), and Ba/F3 HA-TEL-KI-Jak2 subclone 11 (lanes 18 and 19) cells stimulated in the presence (+) or absence (−) of IL-3. EMSAs were performed as described in Materials and Methods. Complexes were resolved on a 5% native polyacrylamide gel. The specificity of DNA binding was determined by the addition of unlabeled β-casein oligonucleotide (S) or a nonspecific oligonucleotide from the DUB-1 promoter (N) and by incubation with a peptide-specific Stat5 antibody. Complexes were analyzed via PhosphorImager detection.