Electrophoretic Mobility-Shift Assay (EMSA)

Nuclear extracts were prepared with a nuclear extract kit (Panomics, Inc., Redwood City, CA) according to the manufacturer's instructions. DNA-binding reactions were performed with equal amounts of nuclear proteins (10 μg) and ³²P-labeled probes at 25°C for 10 min. The following synthetic double-strand oligonucleotide probes were used: Pbx, 5'-CGAATTGATTGATGCACTAATTGGAG-3', and EMX2, 5'-AGGAAGCTGTTTATGTGATCCCCG-3', which have been previously shown to contain the consensus recognition sequences for the HOX-Pbx complexes [23,24]. For cold competition assays, a 100-fold excess of unradiolabeled oligonucleotides was added to the reactions prior to the ³²P-labeled probes. Anti-HOXA7 and anti-PBX2 antibodies used in the supershift experiments were added to the nuclear extract at 25°C for 30 min before the addition of labeled probe. Protein-DNA complexes were resolved in 5% polyacrylamide gel containing 1× TBE (Tris-borate-EDTA: 0.09 M Tris-borate and 2 mM EDTA, pH 8.0). Before loading of the samples, the gel was pre-run for 90 min at 100 V at 4°C. Electrophoresis was carried out at 120 V at 4°C. The gel was then dried under vacuum and exposed to Kodak X-OMAT AR film (Kodak, Rochester, NY).