Anti nrf2 antibody

Immunohistochemical staining was performed on 3 μm paraffinized sections. The samples were cleared in xylene and rehydrated in a series of ethanol washes. Endogenous peroxidase activity was inhibited with 3% H₂O₂ for 10 min and then treated with normal goat serum (1 : 20) for 20 min. Next, the samples were incubated with anti-Nrf2 antibody (mouse monoclonal 1 : 200; Abcam) or anti-8-OHdG antibody (mouse monoclonal 1 : 100; Abcam) at 4°C overnight. The sections were then incubated with a horseradish peroxidase-conjugated secondary antibody (anti-mouse IgG). After rinsing three times with PBS, the sections were stained with 3,3′-diaminobenzidine and then counterstained with hematoxylin. The stained specimens were assessed under a light microscope by a pathologist in a blinded fashion. We randomly selected five high-magnification (×200) fields of the renal corticomedullary boundary zone. The specimens were scored according to the percentage of Nrf2 and 8-OHdG positive cells.

ChIP experiments were performed using the ChIP Assay Kit (Beyotime, China, Cat No.P2078) according to the manufacturer's protocol. Cells were incubated for 10 min in PBS containing 1% formaldehyde and for 5 min in 0.125 M glycine. After centrifugation, cell pellets were resuspended in lysis buffer (1% SDS, 50 mM Tris·HCl pH 8, 10 mM EDTA). Sonication was performed with Bioruptor (Diagenode). Lysates were precleared with 30 μL of protein A-agarose, incubated with 10 μg of anti-NRF2 antibody (Abcam, CA, Cat No. ab62352) or rabbit IgG(Abcam, CA, Cat No. ab172730) overnight at 4°C on rotation, followed by incubation with 30 μL of protein A-agarose for 2 h. After washing the immune complexes were eluted from beads in a buffer containing 1% SDS and 0.1 M NaHCO₃. Cross-linking was reversed overnight at 65°C, and DNA fragments were purified using the QIAquick column (Qiagen, Germany, Cat No. 28106). Quantitative PCRs were performed using 2 μL of DNA in triplicate.

Dulbecco’s modified Eagle’s medium (DMEM), foetal bovine serum (FBS), and other tissue culture reagents were purchased from Gibco BRL Co. (Grand Island, NY). All other chemicals, including lipopolysaccharide (LPS), cobalt protoporphyrin IX chloride (CoPP) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), were obtained from Sigma-Aldrich (St. Louis, MO). Primary antibodies such as anti-iNOS, anti-COX-2, anti-inhibitor kappa B (IкB)-α, anti-p-IкB-α, anti-p50, anti-p65, anti-actin and anti-proliferating cell nuclear antigen (PCNA) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). Anti-p-extracellular signal-regulated kinase (ERK), anti-ERK, anti-p-c-Jun N-terminal kinase (JNK), anti-JNK, anti-p-p38, anti-p38, anti-p-protein kinase B (Akt) and anti-Akt were obtained from Cell Signaling Technology (Danvers, MA). Anti-HO-1 antibody was gained from Merck Millipore (Darmstadt, Germany), and anti-Nrf2 antibody was purchased from Abcam (Cambridge, MA). Anti-mouse, anti-goat and anti-rabbit secondary antibodies were supplied by Merck Millipore (Darmstadt, Germany). Tin protoporphyrin IX (SnPP IX), an inhibitor of HO activity, was obtained from Porphyrin Products (Logan, UT). The enzyme-linked immunosorbent assay (ELISA) kit for PGE₂ was purchased from R&D Systems, Inc. (Minneapolis, MN).

Carnosic acid (Sigma-Aldrich International, Darmstadt, Germany). Indomethacin (Endol 25 mg; 25 cap., DEVA Holding A.S., Istanbul, Turkey); Esomeprazole (Nexium 40 mg; 28 tablets, AstraZeneca Pharmaceutical Company, Istanbul, Turkey) were obtained. ELISA kits measuring IL-1β, IL-6, and TNF-α were obtained from Elabscience, TX, USA. TOS and TAC assays were purchased from Rel Assay Diagnostics, Gaziantep, Turkey. Hematoxylin Eosin (H&E) was obtained from (Merck, Darmstadt, Germany). The following antibodies were utilized: Nrf2 (Anti-Nrf2 antibody, Abcam, Boston, MA, USA, Catalog #: ab31163), HO-1 (Anti-HO-1 antibody, Abcam, Boston, MA, USA, Catalog #: ab13243). Fluorescein-5-Isothiocyanate (FITC; Abcam, secondary antibody, Boston, MA, USA, Catalog #: ab6785) and Texas Red (secondary antibody, Abcam, Boston, MA, USA, Catalog #: ab6719).

The human NSCLC cell lines A549, H460, and H1299 were purchased from the Institute of Basic Medical Sciences Chinese Academy of Medical Sciences & School of Basic Medicine Peking Union Medical College.
Roswell Park Memorial Institute (RPMI) 1640 culture media was obtained from HyClone (Logan, UT, USA). Fetal bovine serum (FBS) was obtained from Gibco (Grand Island, NY, USA). Bicinchoninic acid (BCA), MTT, 2,7-dichlorodihydrofluorescein diacetate(H₂DCF-DA), Giemsa stain, and 4′,6-diamino-2-fenilindol (DAPI) were obtained from Sigma (St. Louis, MO, USA). Brusatol was obtained from Tauto Biotech (Shanghai, China). The anti-Nrf2 antibody and anti-β-Tubulin mouse monoclonal antibody were obtained from abcam. Fluorescein conjugated affinipure goat anti-rabbit IgG (H+L) was obtained from ZSGB-BIO (Beijing, China).
The cell lines A549, H460, and H1299 were maintained in RPMI 1640 (Hyclone) supplemented with 10% FBS and a 100 U/mL penicillin, 100 μg/mL streptomycin solution, and grown at 37 °C in a humidified 5% CO₂ atmosphere.