2.10. Electrophoretic Mobility Shift Assay (EMSA) for NF-κB and STAT3

The NF-κB gel shift oligonucleotide (5′-ACTTGAGGGGACTTTCCCAGGGC-3′) and the STAT3 gel shift oligonucleotide (5′-GATCCTTCTGGGAATTCCTAGATC-3′) were radiolabeled using [³²P]-deoxyadenosine triphosphate (dATP) (Amersham Biosciences, Piscataway, NJ, USA) and T4 polynucleotide kinase (GIBCO, Grand Island, NY, USA). The radiolabeled oligonucleotide was separated from unconsumed [³²P]-dATP using a Bio-Rad purification column (Bio-Rad Laboratories) eluted with Tris-EDTA buffer. Nuclear extracts of the cells were incubated with the [³²P]-labeled oligonucleotide in buffer containing 12% glycerol, 12 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM DTT, 25 mM KCl, 5 mM MgCl₂, 0.04 μg/mL poly[d(I-C)] at room temperature for 30 min. The samples were subjected to electrophoretic separation at 4 °C on a nondenaturing, 5% acrylamide gel. The gel was dried at 80 °C for 2 h after which it was exposed at −80 °C to a radiography film using intensifying screens.