FIG. 5.

(A and B) UV cross-linking of STAT5 complexes to CIS12 probe. Two double-stranded DNA probes comprising CIS1 and CIS2 were used. The first contained 5-bromo-2′-dUTP in regard to the CIS2 binding site (probe A), and the other contained it in regard to CIS1 (probe B) (see Materials and Methods). (A) EMSAs were carried out with UT7 cell extracts stimulated for 15 min. The complexes C1-probe A, C1-probe B, and C0-probe B were cut out after UV irradiation of the gel. (B) These complexes were electrophoresed on an SDS–10% polyacrylamide gel, and the cross-linked complexes were visualized by autoradiography. Molecular masses of the marker proteins are shown in kilodaltons. (C) Effect of the disruption of STAT5 protein interactions by deoxycholate (DOC). EMSA reactions were performed with the CIS12 probe in the presence of purified activated STAT5A (lanes 1 to 4) or STAT5B (lanes 5 to 8). Specific binding of STAT5 proteins to CIS12 probe was determined by supershift assays with specific antibodies directed against STAT5A (lane 1) and STAT5B (lane 8). Different concentrations of deoxycholate were added in the binding reaction mixtures as indicated. DNA-protein complexes were analyzed in nondenaturing acrylamide gels and revealed by autoradiography.