STAT5 Binding Assay Using Biotin-Labeled Probe

Gel shift assay was conducted using a double-stranded, biotin-labeled oligonucleotide probe containing the consensus binding site for STAT5 (sense strand, 5′-AGATTTCTAGGAATTCAATCC-3′), using the Gelshift Chemiluminescent EMSA kit (Active Motif). In brief, protein–DNA complexes were resolved on a nondenaturing polyacrylamide gel, transferred to a positively charged nylon membrane, and cross-linked to the membrane using the UV-light cross-linker. After blocking, the membrane was incubated with blocking buffer containing streptavidin conjugated to HRP. After washing, protein–DNA complexes were detected using a chemiluminescent substrate (Active Motif, Carlsbad, CA, USA)^{40 (link),49 (link)}.