Anti inos

Total protein was obtained using a Protein Extraction Kit (KeyGen BioTECH, Nanjing, China). SDS–PAGE separates proteins of different sizes and electroporates them onto PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% skimmed milk and incubated with anti-ZO-1(ER41204), anti-Occludin(R1510-33), anti-Claudin 1(ER1906-37), anti-Cleaved caspase 3(ET1602-47), anti-Bax(ER0907), anti-Bcl-2(ER1802-97), anti-iNOS(ER1706-89), anti-Arg1(ET1605-8), anti-p-p38(ER2001-52), anti-p38(ET1602-26), anti-p-JNK(ET1609-42), anti-JNK(ET1601-28), anti-p-ERK1/2(ET1603-22), anti-ERK1/2(ET1601-29), and anti-β-actin(R1207-1) antibodies at 4°C overnight(HUABIO, Hangzhou, China). After being washed with Tris Buffered Saline with Tween20 (TBST), the membranes were incubated with secondary antibodies for 1 h. Protein bands were visualized with an enhanced chemiluminescence (ECL) assay kit (Biosharp, Hangzhou, China) and measured with ImageJ software (NIH, Bethesda, MD, USA).

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.