DNA binding of STAT5 in MedB-1 cells. EMSAs were performed using a ⁵²P-labeled β-casein gene promoter region containing the STAT5 DNA binding motif and nuclear protein extracts from MedB-1 cells. Competition with unlabeled probe (lane A) and inhibition of complex formation by anti-STAT5 antibodies (lane B) demonstrate specificity of protein-DNA binding. (Lane C) EMSA on nuclear extracts from MedB-1 cells treated with 20 μM Tyrphostin AG490 indicates a very late effect on STAT5-DNA binding. (Lane D) EMSA on nuclear extracts from MedB-1 cells (none) and cells transfected with wt SOCS-1 (SOCS1) expression plasmid or mock plasmid (mock) for 48 hours. Wild-type SOCS-1 specifically reduces STAT5-DNA binding.