Figure 2

(A) Validation of the STAT5 genomic library by cold competition EMSA analysis. Ten randomly selected STAT5 bound genomic fragments were amplified with M13 primers (upper panel) and used as cold competitors at 30–50-fold molar excess in EMSA assays using IL-2 stimulated YT nuclear extracts and [³²P]-labeled STAT5-probe (lower panel). DNA-binding was expressed as % of control (a reaction without cold competitor (-)) as shown in the graph. The PCR amplicon surrounding the STAT5 binding site in the enhancer of the human CISH gene was used as a positive control. (B) Validation of STAT5 binding to BCL10-SBR. PCR amplified BCL10-SBR was used as a cold competitor in EMSA assays employing IL-2 stimulated YT nuclear extracts and [³²P]-labeled STAT5-probe (a representative of a BCL10-SBR cold competition EMSA analysis is shown). Band intensities were determined by densitometric analysis. The results presented are an average of two independent experiments for BCL10-SBR and three for CISH.