Phospho nf κb p65 antibody

Cells were plated in growth media overnight at a density of 1×10⁶ cells per well on a 6-well plate. Cells were first incubated with 6-methoxyflavone or resveratrol for 12 h and then LPS was added to a final concentration of 500 ng/mL. For the western blot analysis, cells were washed with PBS and then lysed with 150 μL RIPA buffer containing protease and phosphatase inhibitor cocktail. The supernatants were collected by centrifuge at 14,000 g×10 min at 4°C and stored at -80°C. The total protein content of lysates was determined by enhanced BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Electrophoresis was carried out on a NuPAGE Novex Bis-Tris 4–14% gel (Invitrogen, USA) under the reduced condition. After electrophoresis separation, proteins were transferred to membranes and incubated with rabbit monoclonal phospho-NF-κB-p65 antibody (3033S, 1:1000 dilution, Cell Signaling, USA), rabbit polyclonal iNOS antibody (ab3523, 1:500 dilution, Abcam, USA) or mouse monoclonal β-actin antibody (ANT009, 1:1000 dilution AntGene, China). Targeted proteins were then visualized with Qdot 625 conjugate kit (Invitrogen, USA). Gel images were captured with ZF-258 Gel Imaging System (Shanghai Jiapeng Scientific Co. Ltd, China) under illuminating light at 350 nm.

Oxytocin was purchased from Shanghai Hefeng Pharmaceutical Co., Ltd. (No. H31020850, Shanghai, China), and estradiol (No. J20130009, Bayer, Germany) was purchased from Bayer. Both are approved by the State Food and Drug Administration (SFDA). Other reagents and materials included MCC950 (CP-456773, Selleck, USA; CAS number: 210826–40-7; formula: C20H24N2O5S; molecular weight: 404.48 g/mol), mouse PGF_2α kit (production lot number: 516011, Cayman Chemical, USA), mouse PGE₂ kit (production batch number: 514010, Cayman Chemical, USA), BCA protein quantification kit (production lot number: A53225, Thermo, USA), NLRP3 antibody (production batch number: NBP2–12446, Novus Biological, USA), phospho-NF-κB-p65 antibody (production batch number: #3033, Cell Signaling Technology, USA), NF-κB-p65 antibody (production lot number: #4746, Cell Signaling Technology, USA), IL-1β antibody (production batch number: ab9787, Abcam, UK), IL-18 antibody (production lot number: ab191860, Abcam, UK), caspase-1 antibody (production batch number: NBP1–45433, Novus Biological, USA), beta-actin mouse monoclonal antibody (production lot number: #4211, Cell Signaling Technology, USA), goat anti-rabbit secondary antibody (production batch number: AP132P, Merck Millipore, Germany), and goat anti-mouse secondary antibody (production lot number: AP124P, Merck Millipore, Germany).

DSS (AppliChem, Lot#：A3261‐0250 Germany); the six medicinal herbs of QBD raw powder, as shown in Table 1 (Pharmacy Department, The Second Hospital affiliated to Shanxi University of TCM, Xianyang, China) were decoting up 150 mL by traditional decocting method for enema； Mesalazine Enemas (V Vifor AG Zweigniederlassung Medichemie Ettingen， H20150127, Switzerland); faecal occult blood (FOB) (Baso Diagnostics Inc， Zhuhai， china); NF‐κB p65 antibody (Cell Signaling, #8242, Danvers, MA, USA); Phospho‐NF‐κB p65 antibody (Cell Signaling, #3039); p44/42 MAPK (ERK1/2) (antibody (Cell Signaling，#4695); Phospho‐p44/42 MAPK (ERK1/2) antibody (Cell Signaling, #4370); MMP‐9 antibody (Merck, AB19016, Burlington, MA, USA); Ki67 antibody (Merck, AB9260); Cleaved Caspase‐3 antibody (Cell Signaling, #9664); Caspase‐3 antibody (Cell Signaling, #9662); β‐Actin antibody (Cell Signaling,# 4970) Notch1 antibody (Cell Signaling, #3608s); ZO‐1 antibody (abcam, ab96587, Cambridge, MA, USA); Occludin antibody (abcam, ab 21632); claudin‐1 antibody (abcam, ab15098); primer synthesis (Takara Bio Inc, Dalian, China).

MACS-purified CD4⁺CD25⁺ T cells were stimulated with TNF (100 ng/mL), with or without selected inhibitors [SB203580 (SB), Bay 11-7082 (Bay), Sulfasalazine (Sul)] for 30 min. The cells were homogenized in RIPA buffer containing a cocktail of proteinase and phosphatase inhibitors. Protein samples were separated on a SDS-PAGE gradient gel (4–12% Bis-Tris protein gel; Thermo Fisher Scientific) and transferred to PVDF membranes. The blots were blocked with 5% BSA for 1 h and incubated with phospho-p38 antibody (1:1,000; Cell Signaling Technology) and phospho-NF-κB p65 antibody (1:1,000; Cell Signaling Technology) overnight at 4°C. The blots were then incubated in HRP-conjugated secondary antibody (1:3,000) for 1 h at room temperature, developed in ECL solution (Thermo Fisher Scientific) for 1 min, and exposed by G-Box imager. The blots were then incubated in stripping buffer (Thermo Fisher Scientific) at 37°C for 15 min and reprobing with IκBα antibody (1:1,000; Cell Signaling Technology) or p38 antibody (1:1,000; Cell Signaling Technology) or NF-κB p65 antibody (1:1,000; Cell Signaling Technology) or GAPDH antibody (1:3,000; Cell Signaling Technology).

The right lungs of mice were lysed and the protein concentrations were measured by BCA Protein Assay Kit (P0010, beyotime). An equivalent amount of protein samples were separated by 4–15% BeyoGel^™ Plus Precast PAGE (Polyacrylamide gel electrophoresis) Gel (P0465S, beyotime), electrotransferred to PVDF membranes and incubated for 2 h at room temperature with primary antibodies: caspase-3 antibody (9662, Cell Signaling Technology), IL-1β antibody (31,202, Cell Signaling Technology), Phospho-NF-κB p65 antibody (3033, Cell Signaling Technology), NF-κB p65 antibody (BS1253, Bioworld) and GAPDH antibody (60004-1-Ig, Proteintech). Then, the membranes were incubated with secondary antibodies for 1 h at room temperature and exposured with the ChemiDoc XRS + System (Bio-Rad). The results were quantified using ImageJ system (1.52a).

The cells were lysed using radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China) and centrifuged at 4°C for 12,000 rpm. The concentration of the lysate was evaluated using a bicinchoninic acid (Thermo Scientific) protein assay kit. Each sample was diluted with 5Хloading buffer, boiled for 10 min, resolved on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, and then deposited onto polyvinylidene fluoride membranes at equal amounts. The membranes were blocked with 5% skim milk for 1 h and incubated overnight at 4°C with the primary antibodies against β-actin (1:1,000; 5,057; Cell Signaling Technology, Danvers, MA, USA), phospho-NF-κB p65 antibody (1:1,000; 3,033; Cell Signaling Technology), NF-κB p65 (1:1,000; 6,956; Cell Signaling Technology), and MyD88 (1:1,000; NB100-5698SS; RDSC). Proteins were detected using enhanced chemiluminescence detection reagents after being treated with secondary antibodies, HRP-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (Beyotime). All samples were incubated along with β-actin as an internal standard. PEDV anti-nucleocapsid (N) protein antibody (1:1,000) was gifted by Prof. Tong Guangzhi, Shanghai Veterinary Research Institute.

TM6 (Purity > 95%) was purchased from and synthesized by SBS Genetech Co. (Beijing, China). LPS (Escherichia coli 055:B5) was obtained from Sigma (St. Louis, MO, USA). Dexamethasone (Purity > 99%) was provided by Changle Pharmaceutical Co. (Xinxiang, Henan, China). ELISA kits were purchased from Biolegend (San Diego, CA, USA). Phospho-IκBα and, phospho-NF-κB p65 antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA). All other chemicals were reagent grade.