Anti nf κb p65 antibody

RAW264.7 cells were incubated with LPS and different concentrations of GSYJ for 60 min, fixed with 4% paraformaldehyde for 20 minutes, and then, permeabilized with 0.2% Triton-X for 20 minutes. The cells were incubated with the primary anti-NF-κB/p65 antibody (Santa Cruz Biotechnology Inc. Dallas, TX, USA), Alexa Fluor 488-conjugated secondary antibody, and DAPI for 1 hour each at room temperature. Fluorescence was observed under a fluorescence microscope (original magnification, x40; Leica Microsystems, Wetzlar, Germany).

The quantitative analyses of the NOD1, NOD2 and NF-kB protein levels were accomplished with western blot analyses that were performed essentially as previously described [13 (link)]. The rats were killed at the indicated times after infection, and all brain tissue was removed. Protein extraction performed using a Total Protein Extraction Kit and a Nuclear-Cytosol Extraction Kit. The total NOD1 and NOD2 proteins and nuclear NF-κB proteins were prepared. Protein content was measured with a BCA kit according to manufacturer’s instructions. The total protein samples (50 ug) and nuclear protein samples (30 ug) were subjected to electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels (SDS/PAGE) and transferred to nitrocellulose membranes. The membranes were blocked with 5% skimmed milk/TPBS (10 mM Tris–HCl, 150 mM NaCl, 0.05% Tween-20) for 2 h at room temperature and then incubated with anti-NOD1 (1:500, Cell Signaling Technology), anti-NOD2 (1: 500, Cell Signaling Technology) and anti-NF-κB P65 antibody (1 : 500, Santa Cruz Biotechnology). The membranes were washed and incubated with secondary antibody for 1 h, and the relative densities of the bands were then analyzed.

In each group, five animals were sacrificed at T3, and the total protein was extracted from their hippocampal tissues. Protein levels of GABA_B1 receptors, NF-κB, IL-1β and TNFα were analyzed using western blot assay. Briefly, total protein concentrations were determined using bicinchoninic acid assays (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and western blot assays were performed as described above. Primary antibodies included the anti-GABA_B1 receptor antibody (1:500, Santa Cruz Technology, Santa Cruz, CA), the anti-NF-κB p65 antibody (1:500, Santa Cruz Technology, Santa Cruz, CA), the anti-IL-1β antibody (1:500, Santa Cruz Technology, Santa Cruz, CA) and the anti-TNFα antibody (1:500, Santa Cruz Technology, Santa Cruz, CA), as well as the secondary horseradish peroxidase-labeled goat anti-rabbit immunoglobulin (1:4000, PTG Lab, Chicago, IL). The membrane was developed using enhanced chemiluminescence, exposed to X-ray film, and then photographed under UV light using a UVP gel documentation system (UVP, Upland, CA).

For immunohistochemistry examination, kidney sections were immunostained using immunoperoxidase technique with Vector ABC kit (Vector Laboratories). Briefly, sections (3μm thick) were blocked with 3% BSA for 30 min at room temperature, and incubated with primary antibody overnight at 4°C. The primary antibodies used were anti-NF-κB p65 antibody (Santa Cruz Biotechnology mouse monoclonal, 1:200), anti-IL-6 antibody (Santa Cruz Biotechnology rabbit polyclonal, 1:200) and anti-ICAM-1 antibody (Santa Cruz Biotechnology mouse monoclonal, 1:200). Sections were then washed and incubated with biotinylated secondary antibodies for 60 min at room temperature. Biotin was identified and visualized with 3,3’-diaminobenzidine (DAB) solution. As a negative control, the primary antibody was replaced with nonimmune IgG, and no staining occurred. Counterstaining was then performed before examination under a light microscope. Random 100 glomeruli from each renal specimen were observed, and images were then analysed with Image Pro Plus 6.0 edition (Media Cybernetics) for the determination of immunostained area. The percentage of the stained area was calculated as the ratio of suitable binary thresholded image and the total field area.

Indo, LPS (Escherichia coli serotype 0111:B4), λ-carrageenan, and MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) were obtained from Sigma-Aldrich Co. (USA). Prostaglandin E₂ Express ELISA was from Cayman Chemical (USA). TNF-α, IL-1β, and IL-6 ELISA were from Boster Biological Engineering Co., Ltd. (China). Anti-iNOS, anti-COX-2 antibody, and anti-NF-κB p65 antibody were from Santa Cruz Biotechnology (USA). BAY 11-7082 (NF-κB inhibitor) was from Beyotime Institute of Biotechnology (China). The 4-amino-5-methylamino-29,79-difluorofluorescein diacetate (DAF-FM diacetate) was from Invitrogen (USA).

According to the needs of different Co-IP experiments, related proteins were overexpressed through gateway system. Briefly, cell lysates were collected using ice-cold IP lysis buffer. The lysates were transferred to a micro centrifuge tube and centrifuged at 13,000×g for 10 min. The supernatant was transferred to a new tube for protein concentration determination and further analysis. The Co-IP analyses were performed using a Co-Immunoprecipitation Kit (ThermoFisher Scientific, #26149) according to the manufacturer's protocol. The experimental steps including: pre-clear lysate using the control agarose resin, Co-IP, elution of Co-IP, resin regeneration and preparation for SDS-PAGE analysis were carried out in turn. Antibodies used therein include: anti-HDAC5 antibody (Invitrogen, PA1-41117, 1:500), anti-Smad7 antibody (Santa Cruz, sc-365846, 1:200), anti-MEF2A antibody (Santa Cruz, sc-17785, 1:200) and anti-NF-κB p65 antibody (Santa Cruz, sc-8008, 1:200). Normal rabbit IgG without antigenicity provided with the kit was used as a negative control. SDS-PAGE and immunoblotting were performed as described above.