Figure 3.

Induction of STAT6 nuclear binding activity to Iɛ oligonucleotide. (A) Electrophoretic mobility-shift assay. BJAB cells transfected with pcDNA3 vector alone, EpoR, or EpoR/IL-4Rα constructs were left unstimulated (None), or were stimulated for 30 min with Epo (50 units/ml), or IL-4 (200 units/ml). The cells were lysed and nuclear extracts were incubated with ³²P-labeled Iɛ oligonucleotide, then subjected to PAGE. (B) Specificity and identity of the Iɛ oligonucleotide binding nuclear complex. Nuclear extracts from EpoR/IL-4Rα transfectants left unstimulated or stimulated for 30 min with Epo (50 units/ml) or IL-4 (200 units/ml) were incubated with ³²P-labeled Iɛ oligonucleotide and 100-fold molar excess of the following cold competitors: unlabeled Iɛ (Iɛ), Iɛ mutant (Iɛ mut), and an AP-1 consensus sequence (AP-1). For supershift assay, nuclear extracts were pre-incubated with rabbit anti-STAT6 antiserum or with NRS. Similar results were obtained in three independent experiments.