Anti nlrp3

Ultrapure lipopolysaccharide (LPS) (E. coli 0111:B4, cat. no. tlrl-3pelps), standard LPS (E. coli 0111:B4, cat. no. tlrl-eblps), ATP (cat. no. tlrl-atpl), nigericin (cat. no. tlrl-nig), and MSU (cat. no. tlrl-msu) were purchased from InvivoGen, lipofectamine 3,000 transfection reagent (cat. no. L3000015) was purchased from Thermo Fisher, C646 (S7152) was bought from Selleck Chemicals, mouse immunoglobin IgG protein (cat. no. ab198772) was purchased from Abcam, Protein A/G PLUS-Agarose (cat. no. sc-2003) was obtained from Santa Cruz, cell lysis buffer (CLB) (cat. no. 9803) was purchased from Cell Signaling Technology, and mouse IL-1β (cat. no. 88–7013), tumor necrosis factor-α (TNF-α) (cat. no. 88-7324), interleukin-6 (IL-6) (cat. no. 88-701364), and human IL-1β (cat. no. BMS22) ELISA kits were purchased from Thermo Fisher.
Anti–IL-1β (1:1,000, AF-401-NA; RRID:AB_416,684) was purchased from RD System, anti-NLRP3 (1:1,000, Cryo-2) and ASC (1:1,000, AL177) were purchased from Adipogen, anti–caspase-1 (1:1,000, ab179515), and anti-NEK7 (1:5,000, ab133514) were purchased from Abcam; Anti–β-actin (1:10,000, BH10D10), anti-p65 antibody (1:1,000, 8242), anti–p-p65 antibody (1:1,000, 3033), and GAPDH antibody (1:2,000, 5,174) were purchased from Cell Signaling Technology, and DyLight 488–labeled secondary antibody (1:50, A120-100D2) was purchased from InvivoGen.

The protein expressions of HMGB1 and components of inflammasomes were examined by western blotting. Total protein was extracted from the nasal mucosa samples using RIPA lysis buffer and protein concentrations were measured by BCA assay. Protein lysates were separated on 12% Sodium Dodecyl Sulfate (SDS) – Polyacrylamide gels and transferred to Polyvinylidene Difluoride (PVDF) membranes. The membranes were blocked for 1 h with 5% fat-free milk in Tris-Buffered Saline containing Tween (TBST) and incubated overnight at 4 °C with the appropriate dilution of primary antibodies: anti-Nlrp3 (R&D Systems, Minneapolis, MN, USA, diluted 1:300), anti-AIM2 (Abcam, Cambridge, MA, USA, diluted 1:1,000), anti-Asc (Abcam, diluted 1:1,000), anti-caspase-1 (Biovision, Milpitas, CA, USA, 1:200), and anti-β-actin (Millipore, Billerica, MA, USA, diluted 1:3,000). After washing, the membranes were incubated for 1 h at room temperature with the appropriate HRP-conjugated secondary antibody (diluted 1:3,000). The protein bands were visualized by BeyoECL Plus (Beyotime, Haimen, Jiangsu, China), and Gel-Pro analyzer 4.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was used for relative quantification, with β-actin as the internal control.

The FDB or soleus muscles from mice were homogenized by sonication in cold RIPA lysis buffer, containing 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM BAPTA, 20 mM Tris-HCl, pH 7.5, 1% Triton X-100, and Protease Inhibitor Cocktail (Roche Diagnostics, Germany), as described previously [20 (link)]. The samples were loaded onto a 12% SDS-polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA). The primary antibodies used were: anti-NLRP3 (1:500) from R&D Systems (MAB7578, Minneapolis, MN, USA), anti-IL-1β (1:500; SC-32294), anti-caspase-1 (1:500; SC-56036), anti-ASC (1:1000; SC514414), and anti-GSDMD (1:500; SC-393581) from Santa Cruz Biotechnology (Dallas, TX, USA), and anti-GADPH (1:20000) from Sigma-Aldrich (G9545; St. Louis, MO, USA). After washing, the membranes were incubated for 1.5 h with secondary anti-rabbit, anti-rat, or anti-mouse antibodies, as appropriate (Sigma-Aldrich, St. Louis, MO, USA). Western blotting detection reagents (LumiFlash™ Infinity Chemiluminescent Substrate, Taipei, Taiwan) were used following the manufacturer’s instructions, and chemiluminescence was detected using a ChemiDoc Imaging System from Bio-Rad (Hercules, CA, USA). The intensity of the bands was quantified by densitometry, with the use of ImageJ software version 1.44p (NIH, Bethesda, MD, USA).

Protein expression of NLRP3 and caspase-1 was determined using protein analysis of thoracic aortas of in vivo and in vitro all groups. Samples were homogenized in lysis buffer and proteins were collected. Proteins (30 μg) were separated by electrophoresis on 10 or 12% polyacrylamide gels, transferred to 0.22 μm nitrocellulose membranes and blocked using 5% bovine serum albumin (BSA) in Tris buffered saline (TBS) and 0.1% Tween 20 for 1 h. Primary antibodies were incubated overnight at 4°C as follows: anti-NLRP3 (1:500 dilution; R&D Systems), anti-caspase-1 (1:1,000 dilution; Novus Biologicals), anti-β-actin-peroxidase (1:5,000 dilution; Sigma-Aldrich).