Myd88

Briefly, total proteins of each aortic sample were extracted using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing a protease inhibitor (Thermo Fisher Scientific, Shanghai, China). Protein concentration was quantified by the BCA protein assay kit (Beyotime Biotechnology, Shanghai, China), and an equal number of proteins were separated on 12.5% SDS-PAGE gels. After electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes and incubated with antibodies against TLR4 (Wanleibio, Shenyang, China), myeloid differentiation factor 88 (MyD88, Wanleibio), TNF receptor-associated factor 6 (TRAF-6, Wanleibio), interferon regulatory factor 3 (IRF3, Wanleibio), IL-1β (Wanleibio), toll-like receptor-associated activator of interferon (TRIF, Abcam, Cambridge, MA, USA), NF-κB p65 (Abcam), CCL5 (Affinity Biosciences, USA), and β-actin (ZSGB-BIO, Beijing, China). The membranes were blocked with 5% fat-free dry milk and then incubated with the appropriate primary antibody for 24 hours at 4°C. The membranes were then treated with a peroxidase-conjugated secondary antibody after three washes (ZSGB-BIO, Beijing, China). Finally, BeyoECL Plus was used to view the immunological complexes (Beyotime Biotechnology, Shanghai, China).

Jejunum tissues were homogenized at 4°C in RIPA lysis buffer which contained protease inhibitors (PMSF) (Beyotime, Shanghai, China), and concentrations were determined using a BCA assay (35 (link)). Samples were further diluted, and 5× SDS-PAGE loading buffer was added and boiled for 5 min. Equal amounts of protein (10 μg) were loaded onto 12% SDS-polyacrylamide denaturing gels before being transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with tris-buffered saline Tween (TBST) containing 5% non-fat milk powder for 1 h at room temperature, the membrane was incubated overnight with diluted primary antibodies against ZO-1 (1:1,000; ABclonal, Wuhan, China), occludin (1:1,000; Selleck Chemicals, USA), claudin-1 (1:1,000; ABclonal, China), TLR4 (1:500; Proteintech, Wuhan, China), MyD88 (1:500; Wanleibio, Shenyang, China), NF-κB (1:500; Wanleibio, China), NLRP3 (1:1,000; Wanleibio, China), ASC (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), caspase-1 (1:500; Wanleibio, China), IL-1β (1:500; Wanleibio, China), IL-18 (1:1,000; Wanleibio, China), and GAPDH (1:5,000; Proteintech, China). Electrochemiluminescence liquid (ECL) (Tanon, Shanghai, China) was used to detect the signal. ImageJ software was used to assess protein levels. The target protein levels were normalized to GAPDH, and the radioactivity was compared with the control group.

Proteins were extracted from abdominal tissue by lysing it in RIPA lysis buffer and benzyl sulfonyl fluoride (PMSF) for 5 minutes on ice. Following manufacturer’s instructions (Beyotime, China), cytoplasmic proteins and nucleic acids are extracted using nuclear and cytoplasmic protein extraction kits, respectively. An equal amount of protein lysate (30 g per lane) was separated by SDS-PAGE on 10% polyacrylamide gel and transferred to polyvinylidene fluoride membranes. Overnight at 4° C, we used the primary antibodies HMGB1 (cat. no. WL03023, diluted at 1:500, Wanleibio), TLR4 (cat. no. WL00196, diluted at 1:500, Wanleibio), and MyD88 (cat. no. WL02494, diluted at 1:500, Wanleibio). After washing the membrane four times with TBST, the membrane was incubated with the secondary antibody for four hours at 4° C the next day. To quantify the strip strength, Quantity One (Bio-Rad, Shanghai, China) was used as a reference. We standardized the relative protein levels to the same concentration used for the control group.