Anti nrf2

Cell or tissue were lysed or homogenized in a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, and 12 mM Na-deoxycholate supplemented with protease and phosphatase inhibitors cocktail. Nuclei and cytosol were obtained as previously described [26 (link)]. Protein concentration was determined by the Lowry protein assay. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes that were probed with the following antibodies: anti-nNOS (Santa Cruz), anti-SOD1 (Santa Cruz), anti-LDH (Santa Cruz), anti-poly-ADP-ribose polymerase 1 (PARP1), anti-actin (Santa Cruz), anti-tubulin (Sigma), anti-GSNOR (Thermo Scientific), anti-DJ-1 (Santa Cruz), anti-AKT (Santa Cruz), anti-phospho-AKT (Santa Cruz), anti-Nrf2 (Santa Cruz), and anti-H2B (Santa Cruz). After immunostaining with appropriate secondary horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies, bands were revealed using the Amersham ECL detection system.

NRK52E cells grown on coverslips were fixed with 4% paraformaldehyde. Methanol was used for the permeabilization. Cells were washed three times with 0.1% Triton X-100 in phosphate-buffered saline (PBS), incubated overnight at 4 °C in PBS containing 0.1% Triton X-100 and 3% bovine serum albumin to block nonspecific interactions, and then incubated with anti-Nrf2 (Santa Cruz Biotechnology, sc-13032), Plk2 (Santa Cruz Biotechnology, sc-374643), p21^cip1 (Santa Cruz Biotechnology, sc-6246), and p21^cip1 (MyBioSource, San Diego, CA, USA; MBS440016) antibodies. The cells were washed three times with PBST (0.1% Triton X-100) and then incubated with fluorescein isothiocyanate (FITC)–conjugated anti-rabbit secondary antibodies (Invitrogen), fluorescein isothiocyanate (FITC)–conjugated anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories; West Grove, PA, USA), Cy3-conjugated anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories), or Cy3-conjugated anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories) and 4′, 6-diamidine-2-phenylindole (DAPI) (Sigma-Aldrich) for staining nuclear DNA. Images of cells were collected and evaluated with a confocal microscope FW3000 (Olympus; Tokyo, Japan).

Western blot analysis was performed on whole brain with rostral spinal cord tissues harvested 4 h after NTG injection. Cytosolic and nuclear extracts were prepared as described previously [32 (link)]. The expression of nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha (IκB-α), inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), manganese superoxide dismutase (Mn-SOD), and heme-oxygenase 1 (HO-1) was quantified in cytosolic fraction. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf-2) expressions were quantified in nuclear fraction. The filters were probed with specific Abs: anti-NF-κB (1:500; Santa Cruz Biotechnology) or anti-Nrf-2 (1:500; Santa Cruz Biotechnology, SC-722) or IκB-α (1:500; Santa Cruz Biotechnology), anti-MnSOD (1:500; Millipore) or anti-HO-1 (1:500; Santa Cruz Biotechnology), anti-COX-2 (1:500; Cayman) or iNOS (1:500, Santa Cruz) in 1 × PBS, 5% w/v nonfat dried milk, 0.1% Tween-20 at 4 °C, overnight. To ascertain that blots were loaded with equal amounts of proteins, they were also incubated in the presence of the antibody against GAPDH (cytosolic fraction 1:500; Santa Cruz Biotechnology) or lamin A/C (nuclear fraction 1:500 Sigma–Aldrich Corp.) as described [31 (link)].

Nuclear and cytoplasmic proteins were extracted with nuclear and cytoplasmic extraction reagents kits (Pierce, Rockford, IL, USA) according to the manufacturer's protocol for Nrf2, Ho1, and Nqo1 detection. Protein concentrations were measured using BCA protein assay. Proteins were separated by 10% or 12% SDS-PAGE and then transferred onto PVDF membranes. The transferred membranes were blocked with TBS-T (10 mmol/L Tris-HC1, 150 mmol/L NaC1, 0.1% Tween-20) containing 5% skim milk for 1 h at room temperature. The membranes were washed in TBS-T (10 min × 3) and then the membranes were incubated overnight at 4°C with anti-Ho1 (Abcam, Cambridge, UK), anti-Nqo1 (Sigma, St. Louis, USA), anti-Nrf2 (Santa Cruz, CA, USA), anti-β-actin (Abmart, Shanghai, China), and anti-PCNA (Santa Cruz, CA, USA) in TBS-T. The membranes were washed three times again in TBS-T; the membranes were incubated with horseradish peroxidase conjugated to anti-rabbit IgG (Weiao Biotech Ltd, Shanghai, China) overnight at 4°C. The expression of targeted proteins was determined using Odyssey machine and optical density of band was analyzed by using Image J software.

LPS, SB225002, Percoll, and Compound C (CC) were from Sigma-Aldrich (St. Louis, MO). TMZ was from Servier (Tianjin, China). RPMI1640 medium was from Thermo Fisher Scientific (Walham, MA). Nrf2 siRNA was from RiboBio (Guangzhou, China). CXCL2 was from R&D Systems (Minneapolis, MN). DAPI and IL-1β ELISA kit were from Boster (Wuhan, China). Myeloperoxidase (MPO) assay kit and lacate dehydrogenase (LDH) assay kit were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Lipofectamine 2000 was from Invitrogen (Waltham, MA). Protein A/G agroase, anti-Nrf2, and anti-CXCR2 were from Santa Cruz Biotechnology (Dallas, TX). Anti-Ly6G, anti-MPO, anti-GRK2, and isotype antibody were from Abcam (Cambridge, MA). Anti-AMPK and anti-phospho-AMPK were from Cell Signaling Technology (Danvers, MA). Anti-caspase-11 was from Novus Biologicals (Littleton, CO). FITC-CD11b and Percp/Cy5.5-Ly6G were from eBioscience (San Diego, CA). PE-CXCR2 was from BD Biosciences (San Jose, CA). Lysis buffer and BCA protein assay were from Beyotime (Shanghai, China).

To perform experimental tests, all analytical grade reagents were employed. 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), Neocaprione, 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT), PBS (phosphate-buffered saline, pH 7.4), Dimethylsulfoxide (DMSO), and DCFH-DA (2′,7′-Dichlorofluorescin diacetate) were procured from Sigma Aldrich (St. Louis, MO, USA). Dulbecco Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI-1640), and fetal bovine serum were purchased from Gibco, Thermo Fisher Scientific (Waltham, MA, USA). Macrophage Raw 264.7 cells were collected from ATCC (Rockville, MD, USA). Lactate dehydrogenase (LDH) release assay kit was obtained from Cayman Chemical (Ann Arbor, MI, USA). Antibodies, including anti-Superoxide dismutase1 (SOD1), anti-Catalase (CAT), anti-Glutathione peroxidase 1 (GPx-1), anti-HO-1, anti-Nrf2, and β-actin and lamin B were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).