PLoS Med. 2006 October; 3(10): e420.
Nuclear factor erythroid-2 related factor 2 (NRF2), a cap 'n' collar basic leucine zipper transcription factor, regulates a transcriptional program that maintains cellular redox homeostasis and protects cells from oxidative insult, including from chemotherapeutic agents [7–9]. NRF2 activates transcription of its target genes through binding specifically to the antioxidant response element (ARE) found in those gene promoters. The NRF2-regulated transcriptional program includes a broad spectrum of genes, including ones encoding antioxidants (e.g., γ-glutamyl cysteine synthetase modifier subunit [GCLm], γ-glutamyl cysteine synthetase catalytic subunit [GCLc], heme oxygenase-1, superoxide dismutase, glutathione reductase [GSR], glutathione peroxidase, thioredoxin, thioredoxin reductase, peroxiredoxins[PRDX], and cysteine/glutamate transporter [SLC7A11]) [7,8], xenobiotic metabolism enzymes (e.g., NADP[H] quinone oxidoreductase 1 [NQO1], GSTs, and UDP-glucuronosyltransferase) [7,8], and several ATP-dependent drug efflux pumps (e.g., MRP1 and MRP2) [10–12].
Reproductive Biology and Endocrinology 2008, 6:49
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).
