Monoclonal rabbit anti nlrp3 antibody

The following antibodies were used: rabbit anti-NLRP3 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA, dilution 1:1,000); mouse anti-IL-1β monoclonal antibody (Cell Signaling Technology, USA, dilution 1:1,000); mouse anti-Ub monoclonal antibody (Santa Cruz, Dallas, TX, USA, dilution 1:300); rabbit anti-SUGT1 polyclonal antibody (ProteinTech, Chicago, IL, USA, dilution 1:1,000); rabbit anti-HSF1 polyclonal antibody (Cell Signaling Technology, USA, dilution 1:1,000); rabbit anti-pro Caspase-1+p10+p20 monoclonal antibody (abcam, Cambridge, UK, dilution 1:1,000); rabbit anti-IgG (Cell Signaling Technology, USA, dilution 1 μg); mouse antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Cell Signaling Technology, USA, dilution 1:5,000); and mouse anti-ACTIN monoclonal antibody (Cell Signaling Technology, USA, dilution 1:1,000).

Protein lysate samples were separated by 12% SDS-PAGE gel and transferred to the NC membrane for 40 min (100 mM of Tris-HCl, 150 Mm of NaCl, 0.05% Tween 20, and pH 7.2). The NC membrane was blotted with 5% non-fat milk in TBST for 1 h, followed by incubation with a primary antibody diluted with TBST for 2 h at 37°C. The membrane was washed with TBST 3 times and incubated with a secondary antibody at 37°C for 2 h. After washing with PBS 3 times, the NC membrane was stained using a HRP-DAB kit (Zhongshan Golden Bridge, Beijing, China).
The primary antibodies that we used are: mouse anti-p62 monoclonal antibody (Abcam, UK), rabbit anti-LC3A/B monoclonal antibody, rabbit anti-NLRP3 monoclonal antibody, rabbit anti-Caspase-3 monoclonal antibody (Cell Signaling Technology, USA), rabbit anti-caspase-1 p10 monoclonal antibody, and rabbit anti-β-actin monoclonal antibody (Jackson ImmunoResearch, USA). Secondary antibodies were Cy3-conjugated donkey anti-rabbit monoclonal antibody (Jackson ImmunoResearch, USA), HRP-conjugated goat anti-rabbit IgG, and HRP-conjugated goat anti-mouse IgG (Bioworld, USA).

DMSO, LPS (0111:B4), and ATP were purchased from Sigma (St. Louis, MO). RIPA lysis buffer and Nitric Oxide Assay Kit were obtained from the Beyotime Institute of Biotechnology (Shanghai, China). Rabbit anti-NLRP3 monoclonal antibody was purchased from Cell Signaling Technology (Beverly, MA). Rabbit anti-ASC polyclonal antibody and rabbit anti-caspase-1 polyclonal antibody were from Santa Cruz Biotechnology Inc (Santa Cruz CA, USA), and mouse anti-β-actin monoclonal antibody was from Protein Tech Group (Chicago, IL). Micheliolide (MCL) (Molecular Weight: 248.3, purity >99%) was isolated from the Michelia compressa (Magnoliaceae) described previously [34 (link)]. Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) (04-001-1A) were obtained from HyClone Laboratories Inc (Logan, UT, USA) and Biological Industries (BI, IL), respectively. Middle brook 7H9 media were obtained from Difco (Detroit, MI, USA), and oleic acid-albumin-dextrose-catalase (OADC) supplements were from BD Biosciences (BD, Sparks, MD, USA).