2.6. Electrophoretic mobility shift assay (EMSA)

Oligonucleotides containing the consensus sequence of STAT1 (5′-CAT GTT ATG CAT ATT CCT GTA AGT G-3′) or STAT3 (5′-GAT CCT TCT GGG AAT TCC TAG ATC-3′) were end-labeled with (³² P) ATP using T4 polynucleotide kinase and purified using Chroma Spin-10 columns. Samples were incubated with labeled oligonucleotide (20,000–30,000 cpm) for 20 min at room temperature in 1X binding buffer (5X binding buffer: 50 mM Tris-HCl buffer, pH 7.5, containing 20% (v/v) glycerol, 5 mM MgCl₂, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl and 0.25 mg/ml poly(dI-dC)). Products were separated by electrophoresis in a 6% (w/v) non-denaturing polyacrylamide gel using 0.5X TBE (Tris/borate 45 mM, EDTA 1 mM) as the running buffer. Gels were dried and the radioactivity quantitated in a Phosphoimager 840 (Amersham Pharmacia Biotech. Inc., Piscataway, NJ).