Anti nf κb p65

Protein from tissues of spinal dorsal horn was extracted in RIPA lysis buffer, which was bought from Beyotime Biotechnology of China. Through BCA Protein Assay (Thermo Fisher Scientific), the protein concentration was assessed. To separate 30 μg of protein samples, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was then utilized. After separation, protein samples were placed in a polyvinylidene difluoride (PVDF) nitrocellulose membrane (Millipore, Boston, MA, United States), arrested by 5% fat-free dry milk at 25°C in TBST for 1 h and cultured at 4°C with primary antibodies overnight. Primary antibodies included the following: anti-ASC (1:500; ABclonal), anti-Nlrp3 (1:1000; Proteintech), anti-GSDMD (1:1000; Abcam), anti-cleaved Caspase-1 (1:1000; Proteintech), anti- NF-κB/p65 (1:2000; Abcam), and anti-GAPDH (1:10,000; Cell Signaling Technology, CST). The membranes were cultured with a rabbit or mouse HRP-conjugated secondary antibody (1:2000; Beyotime Biotechnology) at 25°C for 1 h after being washed in TBST. Finally, the densities of the target proteins relative to that of GAPDH were quantified using Image Pro Plus software.

Five sections of the brain cortex and anterior horns of the spinal cord from each rat were randomly selected and subjected to immunofluorescence staining. The tissue sections were incubated overnight at 4 °C with anti-NF-200 (1:500; Abcam, Cambridge, MA, USA), anti-GAP-43 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-activated caspase-3 (1:500; Cayman Chemical, Ann Arbor, MI, USA), anti-BDNF (1:500; Abcam), anti-NF-κB p65 (1:500; Abcam), Anti-p75NTR (1:500; Abcam), anti-MBP (1:500; Abcam), anti-glial fibrillary acidic protein (GFAP, 1:200, Thermo Fisher Scientific, Waltham, MA, USA), anti-COX-2 (1:1000; BioVision, Milpitas, CA, USA), and anti-CD68 (1:100; Santa Cruz Biotechnology) antibodies, individually. After washing in PBS, the sections were incubated with TRITC/FITC-conjugated secondary antibodies (1:200; Invitrogen, Carlsbad, CA, USA) for 1 h at 37 °C. The sections were subsequently mounted on glass slides and coverslipped with Antifade Gel Mount Aqueous Mounting Media (Southern Biotech, Birmingham, AL, USA). Stained sections were viewed under a microscope at 200x magnification, and images were taken of three fields per tissue section. Areas stained positively for GFAP, MBP, and CD68 were analyzed using NIH Image software. The numbers of cells stained positively for GAP43, caspase-3, NF-200, BDNF, NF-kB p65, COX-2, Caspase-3, and p75NTR were counted.

Mucosa specimens were rapidly scraped from underlying gastric tissue layers using
two glass slides, which were kept on ice. The mucosal tissues were weighed,
minced by ophthalmic scissors, and homogenized in radioimmunoprecipitation assay
(RIPA) lysis buffer containing protease and phosphatase inhibitor mixture (1%
phenylmethanesulfonyl fluoride (PMSF) and cocktail; Beyotime). The total protein
concentration was measured using a BCA protein assay kit (Beyotime) and a BioTek
microplate reader. Protein extracts (30 µg) were separated with 10% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then the
proteins were transferred onto polyvinylidene difluoride (PVDF) membranes
(Millipore, USA). The blots were blocked with 5% skim milk in Tris-buffered
saline Tween-20 (TBST) for 1 h and subsequently probed overnight at 4°C with
rabbit polyclonal anti-NF-κB p65 (1:2000) primary antibodies (Abcam, UK). Then,
the blots were washed with Tris-buffered saline containing Tween-20 and
incubated with a goat anti-rabbit horseradish peroxidase (HRP)-conjugated
secondary antibody (Abcam) for 1.5 h at room temperature. The bands were
observed using an enhanced chemiluminescence substrate, and protein expression
was quantified using a fully automated image analysis system (Tanon 4500 s;
Tanon Technology Co., Ltd., China).

Lysis of jejunal tissue was prepared using lysis buffer (Sigma, Saint Louis, MO, USA). Total protein concentration was determined using the BCA method. Western blotting analysis was performed as described in a previous study [18 (link)]. The primary antibodies included rabbit anti-iNOS (HuaBio, Hangzhou, China), anti-ERK (Cell Signaling Technology, MA, USA), anti-phospho-ERK (Cell Signaling Technology, MA, USA), anti-p38 (Cell Signaling Technology, MA, USA), anti-phospho-p38 (Cell Signaling Technology, MA, USA), anti-JNK (Cell Signaling Technology, MA, USA), anti-phospho-JNK (Cell Signaling Technology, MA, USA), anti-NF-κB-p65 (Abcam, Cambridge, UK), anti-TRAF6 (HuaBio, Hangzhou, China), anti-HO1 (HuaBio, Hangzhou, China), and anti-GADPH (HuaBio, Hangzhou, China). The second antibody was HRP, goat anti-rabbit IgG, and goat anti-mouse IgG (HuaBio, Hangzhou, China).
Protein bands were visualized with a chemiluminescence substrate (Millipore, MA, USA) and a gel-imaging system (Tanon Science and Technology, Shanghai) and analyzed with Image-J Analysis software (NIH, Bethesda, MD, USA). As an internal control, GADPH showed no difference among the three groups. In all cases, the density values of bands were corrected after subtracting background values. GAPDH was used as an internal reference protein.

After treating with ACAP for 1 h, LPS (2 μg/ml) was added to stimulate RAW 264.7 cells for 24 h. The protein of cells was extracted and then separated on SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes, which were blocked in 5% bovine serum albumin for 1 h at room temperature and incubated overnight. Antinuclear factor-κB (IκB) alpha, anti-IκB alpha (phospho S36), anti-NF-κB p65, and anti-NF-κB p65 (phospho S536) were purchased from Abcam (Shanghai, China) for the immunoblotting, and the relative protein expression was quantified compared with the β-actin level.

Bovine serum albumin (BSA) was obtained from Shanghai Hengyuan Technology Biology Co., Ltd (Shanghai, China). Glucose, Phenylmethylsulfonyl Fluoride (PMSF), and ethylene diamine tetraacetic acid disodium salt (EDTA) were obtained from Beijing solabo Technology Co., Ltd. (Beijing, China). Penicillin and streptomycin were bought from Sangon Biotech Inc. (Shanghai, China). Ethanol (purity > 99%) was obtained from Xilong Chemical Co., Ltd. (Guangdong, China). Fetal bovine serum (FBS) was bought from Sangon Biotech, Inc. (Shanghai, China) and Dulbecco’s modified eagle medium (DMEM) was purchased from ThermoFisher (MA, USA). CCK-8 kit was obtained from Shanghai Yanjin Biotechnology Co., Ltd. (Shanghai, China). Anti-p-P38, anti-P38, anti-JNK, anti-p-JNK, anti-NF-κB p65, anti-p-NF-κB p65, anti-IκB, anti-p-IκB, anti-AKT and anti-p-AKT antibodies were purchased from Abcam Technology (Cambridge, UK). Anti β-actin antibody, Goat anti-rabbit and mouse IgG and mouse anti-goat secondary antibodies were purchased from ZSGB Biotech Co., Ltd. (Beijing, China). Unless specified, all other reagents are obtained from Sigma Chemical Co. (St. Louis, MO, USA).