Myd88

Total protein was extracted from nasal mucosa using RIPA lysis buffer and PMSF. Protein concentration was determined using the Bicinchoninic acid (BCA) Protein Assay kit (Solarbio, Beijing, China). Equal amount of protein (10–20 µg) from each sample was isolated using SDS-PAGE (8%, 12%, and 15% gel) and transferred to the polyvinylidene difluoride (PVDF) membranes. After blocking with 5% nonfat milk for 1 h at room temperature, the membranes were incubated with primary antibodies at 4°C overnight, followed by the incubation of horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3,000, Solarbio) for 1 h at 37°C. Subsequently, the membranes were visualized with electrochemiluminescence (Solarbio) for luminescence generation. GAPDH served as inner control for normalization. The primary antibodies used in present study were: the nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3, 1:1,000, ABclonal, Wuhan, China), apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC, 1:1,000, ABclonal), cleaved caspase-1 (1:500, ABclonal), IL-1β (1:1,000, ABclonal), TLR4 (1:1,000, ABclonal), MyD88 (1:1,000, ABclonal), p65/p-p65 (Ser536) (1:1,000, Affinity, Cincinnati, OH, USA), GAPDH (1:10,000, Proteintech, Wuhan, China), and Histone H3 (1:1,000, GeneTex, San Antonio, TX, USA).

Total, cytosolic, and nuclear proteins were extracted from tissues or cell samples using a total protein extraction kit and a nuclear/cytoplasmic extraction kit (Beyotime, China). Western blot analysis was performed as previously described, with some modifications.^{25 (link)} Proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes, which were blocked in 5% nonfat milk for 2 h at 37 °C and incubated in the presence of primary antibodies (1:1000 dilution) overnight at 4 °C. The following day, the membranes were washed 4 times with 1× Tris-buffered saline containing 0.5% Tween-20 and incubated with secondary antibodies for 4 h at 4 °C. Finally, all membranes were washed 4 times, after which the blots were visualized using enhanced chemiluminescence (Beyotime). Total protein was used to determine TLR4, MyD88, RAGE, NLRP3, GSDMD/GSDMD-N, caspase-1/cleaved caspase-1, and caspase-11/cleaved caspase-11 expressions (ABclonal Technology Co, Ltd, Wuhan, China). The expression of the NF-κB/P65 (CST, Inc, MA) subunit was measured in cytosolic and nuclear proteins. The Image-Pro Plus software (version 6.0) was used for protein band quantification.