Ep1808y

The following antibodies were used for immunoblotting: rabbit polyclonal C-20 (Santa Cruz, CA), mouse monoclonal (M01) clone 1 F3 (Abnova, Taiwan), and rabbit monoclonal EP1808Y (Abcam, Cambridge, UK) for Nrf2; Actin was from Calbiochem/MerckMillipore (Watford, UK); NQO1 was from Novus Biologicals (Cambridge, UK); G6PD was from Bethyl (Montgomery, TX); HIF-1α was from BD Biosciences (San Jose, CA); Cleaved PARP, total AKT, phosphorylated AKT (Ser473) (p-AKT), total ERK1/2, phosphorylated ERK1/2 (Thr202/Tyr204) (p-ERK), Cul3, Keap1, HSP90 and Lamin A/C antibodies were all from Cell Signaling Technology (Danvers, MA); GAPDH was from Advanced Immunochemical Inc. (Long Beach, CA); Secondary antibodies were from DAKO.
N-acetyl-L-cysteine, ascorbic acid, tert-butylhydroquinone, camptothecin, etoposide and staurosporine were all obtained from Sigma (Dorset, UK).

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.