Anti nlrp3 antibody

Total protein was extracted from glomerular mesangial cells using a protein extraction kit (Kaiji, Shanghai, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). Immunoblotting was performed using anti-NLRP3 antibody (rabbit; 1 : 4,000; Abcam), anti-TXNIP antibody (rabbit; 1 : 800; Abcam), anti-procaspase-1 antibody (rabbit; 1 : 700; Cell Signaling Technology), and anti-β-actin antibody (mouse; 1 : 3,000; Beyotime).

Mouse maxillae were fixed in 10% formalin, decalcified in 10% EDTA, embedded in paraffin, and cut into serial sections (5 μm) for immunohistochemical staining. The primary antibodies anti-VDR (1:100), anti-AhR (1:200), anti-CYP1A1 (1:200), anti-p-p65 (1:200), anti-NLRP3 (1:200), anti-ASC (1:200), anti-caspase-1 (1:200), anti-IL-1β (1:100), and anti-IL-6 (1:100), and the secondary antibodies (1:1000) were incubated with the section. All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), except the anti-NLRP3 antibody (Abcam, Cambridge, MA, USA). Mean optical density of the staining was calculated using Image-Pro Plus software (Version 6.0, Media Cybernetics, Silver Spring, MD, USA), and the measurements obtained from both sides were averaged to represent each sample.¹¹ (link)

Main instruments: Vernier caliper (Meinaite, Germany), Ultrasound imaging equipment (Vevo 3100), dehydrator, embedding machine, slicer, spreader (Leica, Germany), automatic tissue scanner (VS120, Olympus).
Main reagents: Cynarin (Chengdu Pufei De Biotechnology, 17041106), xylene (Sinopharm, 10023418), Ethylenediamine tetraacetic acid (Sinopharm. 10009617), 75%Ethanol (Sinopharm, 80176961), Absolute ethanol (Sinopharm, 10009218), Hematoxylin(sigma, H3136), Eosin Y (sigma, E4009), Monosodium Urate (sigma, U2875), Bovine Serum Albumin (Aladdin, B265994),Anti-F4/80 antibody (Abcam, ab6640), Macrophage-stimulating factor (MCSF) (Peprotech, 315–02), Anti-iNOS antibody (Abcam, ab3523), VECTASHIELD Mounting Medium with DAPI (Vectorlabs, H-1200), Trypsin antigen repair solution (Leagene Biotechnology, IH0310), Cell Counting Kit-8 (Dojindo laboratories, CK04), PrimeScript™ RT Reagent Kit (TaKaRa, RR037A), qPCR SYBR Green Master Mix (YESEN, 11201ES03), p-p65 antibody (CST, 3033), p65 antibody (CST, 8242), GAPDH antibody (sigma, G9545), p-p38 antibody (CST, 4511), p-IKKa/β antibody (CST, 2697),Anti-rabbit antibody (CST, 7074), p-JNK antibody (CST, 4668), p-ERK1/2 antibody (CST, 4370), Caspase 1/p20/p10 antibody (Proteintech, 22915-1-AP), Anti-NLRP3 antibody (Abcam, ab263899), Anti-IL-1β antibody (Abcam, ab200478).

Ileum tissues were homogenized, and the resulting total protein was extracted by RIPA lysis mixed with PMSF (Solarbio, Beijing, China). Then, 50 μg of protein per sample was subjected to 7.5%, 10%, or 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Solarbio, Beijing, China). After overnight incubation at 4°C with anti-NLRP3 antibody (dilution at 1 : 1000, Abcam, Cambridge, UK), anti-caspase-1 antibody (dilution at 1 : 300, Santa Cruz, Oregon, USA, SC-398715), anti-IL-1β antibody (dilution at 1 : 600, Bioss, Beijing, China), anti-IL-18 antibody (dilution at 1 : 600, Bioss, Beijing, China), anti-β-actin antibody (dilution at 1 : 2000, Servicebio, Wuhan, China), and anti-HSP-90 antibody (dilution at 1 : 1000, Santa Cruz, Oregon, USA), the membranes with blotted proteins were then incubated with HRP-conjugated goat anti-rabbit secondary antibody (dilution at 1 : 2000, CST, Boston, USA) or rat anti-mouse secondary antibody (dilution at 1 : 2000, Servicebio, Wuhan, China) for an hour at room temperature. After washing three times with TBST, the electrochemiluminescence solution (ECL, Millipore, Massachusetts, USA) was added to the membranes, and then, the membranes were exposed to the exposure machine (ChemiScope series, Clinx Science Instruments Co., Ltd), and the resulting images were recorded and analyzed.

Primary antibodies, such as rabbit anti-NFκB p65, rabbit anti-p-NFκB p65 (Ser536), and rabbit anti-β-actin antibodies, were obtained from Cell Signaling Technology (USA). Anti-GSDMDC1 (64-Y), anti-IL-1β, anti-Hsp60, HRP-labeled goat anti-rabbit IgG, and HRP-labeled goat anti-mouse IgG were purchased from Santa Cruz Biotechnology (USA). The anti-NLRP3 antibody was acquired from Abcam (Cambridge, UK). The IL-1β Quantikine ELISA kit was from MultiSciences (China). The ROS assay kit, N-acetyl-cysteine (NAC) and Cell Counting Kit-8 (CCK-8) were from Beyotime Biotech (China). IKK-16 was purchased from Selleck Chemicals (USA). Sodium iodide (NaI) was purchased from Sigma-Aldrich.