Anti aim2

The following antibodies were used for immunoblot: anti-β-actin, anti-ASC, anti-HO-1, anti-HO-2, anti-ATF4, and anti-CHOP antibodies were purchased from Cell Signaling Technology. Anti-NLRP1, anti-NLRP3, anti-NLRC4, anti-AIM2, and anti-pro-caspase-1 antibodies were purchased from Abcam. Anti-caspase-1 antibody (against both pro-caspase-1 and p20) was purchased from Santa Cruz Biotechnology. Anti-IL-1β (against both pro-IL-1β and mature IL-1β) was purchased from Novus Biologicals.

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.