Anti cox 2

Lung samples were homogenized in RIPA buffer (AR0101-100, Boster, Wuhan, China) with a protease inhibitor cocktail. The protein concentration was measured by the BCA method (Thermo Scientific, MA, USA). Samples containing 30 μg of denatured total protein were separated by 10% SDS-PAGE and then transferred to 0.45 μm PVDF membranes. The membranes were blocked with 5% nonfat dry milk for 1 hour and incubated overnight at 4°C with the following antibodies: anti-ICAM-1 (dilution, 1 : 1000, Abcam, Cambridge, Massachusetts, USA), anti-P65 (dilution, 1 : 1000, CST, USA), anti-IκBα (dilution, 1 : 2000, HuaBio, Hangzhou, China), anti-p-P65 (dilution, 1 : 1000, CST, USA), anti-COX-2 (dilution, 1 : 1000, HuaBio, Hangzhou, China), and anti-β-actin (dilution, 1 : 1000, Bioss, Beijing, China). HRP-conjugated secondary antibodies (dilution, 1 : 5000, BA1054, Boster, Wuhan, China) were incubated for 1 hour at room temperature. Then, proteins were detected by chemiluminescence reagent (AR1171, Boster, Wuhan, China). The gray value of the band was observed and analyzed using the ImageJ software.

RIPA buffer was used to extract total protein from THP-1 cells or foot tissues. Cytosolic protein was extracted by Cytoplasmic Protein Extraction Kit (Beyotime, China). Nuclear protein extraction was performed using the CellLytic™ NuCLEAR™ Extraction Kit (Sigma, USA). The protein concentrations were measured using a BCA protein assay kit (Thermo Scientific, MA, USA). The protein samples were denatured by SDS-PAGE and transferred onto PVDF membranes. After blocking, the membranes were incubated with the primary antibodies at 4 °C overnight; anti-Phospho-NF-kB p105/50(Ser933) and anti-Phospho-NF-κB p65(Ser536) were obtained from Cell Signaling Technology (CST, USA); and anti-IκBα, anti-COX-2, anti-SOD2, anti-MyD88, anti-TLR4, anti-Caspase-1, anti-Lamin B1, anti-IL-1β, anti-NF-kB p105/50, anti- NF-κB p65, and anti-NLRP3 were obtained from HuaBio (Hangzhou, China). Then, the membranes were incubated with the secondary antibodies (1:5000) for 1 h at room temperature and exposed to the gel imaging system with a chemiluminescence kit. The band intensity was quantified using ImageJ software.

RIPA buffer was applied to extract total protein from either macrophages or foot pad tissues. Cytoplasmic and Mitochondrial Proteins were extracted using Cytoplasmic and Mitochondrial Protein Extraction Kit (Sango Biotech, Shanghai, China) following the manufacturer's instruction. Cell culture supernatants were collected and concentrated (×10) using Amicon Ultra Centrifugal Filters (MilliporeSigma). Proteins were suspended in Laemmli Buffer 1×, boiled at 95°C for 6 minutes, and resolved by SDS-PAGE. Then, proteins were transferred to PVDF membrane. Western blot was performed using the following antibodies: anti-IL-1β, anti-Phospho-NF-κB p65(Ser536), and anti-Phospho-NF-κB p105/50 (Ser933) were purchased from Cell Signaling Technology (CST, USA), and anti-COX-2, anti-iNOS, anti-MPO, anti-IκBα,, anti-MyD88, anti-Caspase-1, anti-TLR4, anti-NF-κB p65, anti-NF-κB p105/50, and anti-NLRP3 were obtained from HuaBio (Hangzhou, China). The membrane was incubated with the primary antibodies at 4°C overnight. After washing the membrane, the membrane was incubated with second antibodies (1 : 7500 dilution, Boster, Wuhan, China) for 1 h at room temperature. Finally, the membrane was exposed to the gel imaging system with a chemiluminescence kit. The band intensity was quantified using Image J software.