Mouse anti nf κb

Cells were seeded in 24‐well plates over coverslips (1 × 10³ cells/well) and treated as described in Figure legend. Then, cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X‐100 in PBS, blocked with 3% BSA/PBS and stained with primary antibodies: rabbit anti‐Ki67 (Abcam), mouse anti‐NF‐κB (Santa Cruz Biotechnology), mouse anti‐β‐catenin (Santa Cruz Biotechnology), mouse anti‐NANOG, rabbit anti‐Oct‐4 and mouse anti‐SOX‐2 (Cell Signaling Technology, Danvers, MA, USA). Samples incubated in 3% BSA/PBS only represented negative controls (Supplementary Figure S3). The corresponding FITC‐coupled secondary antibodies (Sigma‐Aldrich) and 1 ng/mL of nuclear dye DAPI (Sigma‐Aldrich) were added during 2 hours. Samples were examined using an epi‐fluorescent microscope (Olympus, Japan).

Ferulic acid and 5-hydroxymethyl-2-furaldehyde (5-HMF) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Albiflorin and paeoniflorin were the products of Wako (Osaka, Japan). Nodakenin was purchased from NPC BioTechnology Inc. (Daejeon, Korea). The purity of all reference standards was ≥98.0%. HPLC-grade methanol, acetonitrile, and water were obtained from J.T.Baker (Phillipsburg, NJ, USA). Glacial acetic acid, analytical reagent grade, was purchased from Junsei (Tokyo, Japan). RPMI 1640, fetal bovine serum, TNF-α, tissue culture reagents, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxy-methylester (BCECF-AM), DAF-FM diacetate, and CM-H₂DCFDA, Alexa Fluor 488 and 594 conjugated second antibodies were purchased from Invitrogen (San Diego, CA). Biotin 3′ End DNA Labeling Kit, LightShift® Chemiluminescent EMSA Kit, Biodyne® Precut Nylon Membranes, Lipofectamine LTX reagent, and Renilla-Firefly Luciferase Dual Assay Kit were purchased from Pierce Biotechnology (Rockford, USA). Primary antibodies, including mouse anti-ICAM-1, goat anti-VCAM-1, rabbit anti-E-selectin, mouse anti-NF-κB, mouse anti-p-IκB-α, rabbit anti-HO-1, and rabbit anti-Nrf2, were purchased from Santa Cruz Biotechnology (CA, USA). Donkey anti-goat IgG-H+I were purchased from Bethyl (Montgomery, USA) and goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Enzo (Farmingdale, USA).

For in vivo studies, spinal cord segments 7 days after surgery were fixed and prepared accordingly. Sections were incubated in 3% H₂O₂ for 15 min. at room temperature following by 30 min. incubation in 5% albumin from bovine serum in PBS containing 0.1% Triton X‐100 in a 37°C oven. After blocking, sections were incubated with rabbit anti‐cleaved caspase 3 (1:400; Cell Signal Technology, Danvers, MA, USA), goat anti‐Iba‐1 (1:400; Abcam, Cambridge, MA, USA), mouse anti‐NeuN (1:400; Abcam) or mouse anti‐TLR4 (1:400; Abcam) at 4°C overnight. After primary antibody incubation, sections were washed at room temperature and were incubated with appropriate secondary antibodies for 1 hr. The nuclei were stained with DAPI 19, 20. For in vitro studies, cells were washed in PBS, and fixed in 4% PFA for 30 min. at room temperature following by 30‐min. incubation in 5% bovine serum albumin at 37°C. Sections were stained with primary antibodies at appropriate dilutions (rabbit anti‐cleaved caspase 3 (1:400), goat anti‐Iba‐1 (1:400), mouse anti‐TLR4 (1:400) or mouse anti‐NF‐κB (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) followed by incubation with appropriate secondary antibodies 21. Images were captured using a fluorescence microscope (Nikon,Tokyo,Japan) and confocal microscopy (Nikon,Tokyo,Japan).