Complementary dna oligonucleotides

Complementary DNA oligonucleotides (Integrated DNA Technologies) were resuspended in a buffer consisting of 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl, mixed at equimolar concentrations, and then hybridized by heating to 95 °C for 5 min, followed by cooling slowly to room temperature. DNA oligonucleotides used in EMSA and single-wavelength analytical ultracentrifugation experiments were FAM-5ʹ labeled on both strands to improve sensitivity. Double-stranded DNA oligonucleotides were stored at −20 °C until use.

EMSA was performed using the LightShift Chemiluminescent EMSA kit (Pierce Biotechnology, Rockford, IL, USA) according to manufacturer’s instructions. Briefly, nuclear protein extract was isolated from brain microvessels prepared from Nx, Hx, H/R, and sulforaphane treated rats. Complementary DNA oligonucleotides 5′-CGG TCA CCG TTA CTC AGC ACT TTG-3′ and 5′-CAA AGT GCT GAG TAA CGG TGA CCG-3′ (antioxidant response element recognition sequence highlighted) were purchased from Integrated DNA Technologies, end labeled with biotin, and annealed at 95 °C for 5 min. EMSA samples were prepared using 5 μg of nuclear extract in each Nrf2/ARE binding reaction. The binding reaction was incubated at room temperature for 15 min and DNA–protein complexes were resolved on a precast 6% native polyacrylamide gel in 0.5% TBE buffer. The gel was removed from the electrophoresis unit, blotted onto a nitrocellulose membrane, and incubated with streptavidin-horseradish peroxidase for 30 min. Membranes were developed using enhanced chemiluminescence. Experiments to determine specificity of EMSA reactions for the Nrf2/ARE complex were conducted by incubating binding reactions in the presence of a rabbit monoclonal anti-Nrf2 antibody (EP1808Y; 1/20 dilution; Abcam, Cambridge, MA). Control EMSA experiments were performed by adding an excess (200×) of unlabeled probe to binding reactions.