Anti il 1β rabbit monoclonal

The PVN tissues were homogenized in RIPA lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China) containing PMSF (Beyotime Institute of Biotechnology) (ratio of 100:1). A BCA assay kit (Pierce Protein Biology, St. Louis, MO, USA) was used to determine the protein concentration. Approximately 70 μg of total protein for each sample were resolved on 8%‐15% polyacrylamide gels and transferred onto PVDF membranes (Bio‐Rad, Richmond, VA, USA). The membranes were blocked for 1 hour using 5% nonfat dry milk diluted with TBS and then probed with different antibodies overnight at 4°C: anti‐SAP130 rabbit monoclonal (Abcam; 1:2000), anti‐Mincle rabbit polyclonal (Bioss corporation; 1:1000), anti‐NLRP3 rabbit polyclonal (Abcam; 1:500), anti‐IL‐1β rabbit monoclonal (Cell Signaling Technology, Danvers, MA, USA; 1:1000), anti‐Iba‐1 goat polyclonal (Abcam; 1:1000) and anti‐GAPDH rabbit polyclonal (Goodbio Technology, Wuhan, China; 1:1000) antibodies. Following a 2 hours‐incubation with HRP‐conjugated anti‐rabbit or anti‐goat secondary antibodies (GenScript, Piscataway, NJ, USA; 1:5000), and immunodetection was conducted using the ECL (Millipore, Billerica, MA, USA) method. NIH ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to analyse the grey scale value.

Nigericin and anti-β-actin (mouse) monoclonal antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). LPS was prepared by A. actinomycetemcomitans as described previously [25 ,26 (link)]. LY294002 was purchased from MedChem Express (Monmouth Junction, NJ, USA). The JNK Inhibitor II and PD98059 were purchased from Merck Millipore (Billerica, MA, USA). SB239063 was purchased from Calbiochem (San Diego, CA, USA). Mg(OH)₂ nanoparticles were prepared in the same way as previously described [20 (link)]. Anti-IL-1β (rabbit) monoclonal, anti-IκBα (rabbit) polyclonal, anti-NF-κB p65 (rabbit) monoclonal, anhosphorpho-p38 MAPK (rabbit) monoclonal, anti-p38 MAPK (rabbit) polyclonal, anti-phospho-ERK1/2 (rabbit) monoclonal, anti-ERK1/2 (rabbit) monoclonal, anti-phospho-JNK (rabbit) polyclonal, anti-JNK (rabbit) polyclonal, anti-phospho-Akt (rabbit) monoclonal, and anti-Akt (rabbit) polyclonal antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). An anti-β-actin (mouse) monoclonal antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). The anti-lamin B1 (rabbit) polyclonal antibody was purchased from Proteintech Biotechnologies (Rosement, IL, USA).