Stat1 and Stat3 DNA binding is constitutively activated in TEL-Jak2 transformed Ba/F3 cells. Nuclear extracts were prepared from Ba/F3 (lanes 1 through 7) and Ba/F3 HA-TEL-Jak2 JH1 subclone 36 (lanes 8 through 14) cells stimulated in the presence (+) or absence (−) of IFN-. EMSAs were performed as described in Materials and Methods using a ³²P-labeled oligonucleotide from the IRF-1 promoter. Complexes were resolved on a 5% native polyacrylamide gel. The specificity of DNA binding was determined by the addition of unlabeled IRF-1 oligonucleotide (S) or a nonspecific oligonucleotide from the DUB-1 promoter (N) and by incubation with peptide-specific Stat1 (S1), Stat3 (S3), or Stat5 (S5) antibodies. Complexes were analyzed via PhosphorImager detection.