Nlrp3

Antigen retrieval was performed on paraffin sections (3–5 μm) by boiling in citrate antigen retrieval buffer (pH 6.0) or EDTA buffer (pH 8.0), followed by blocking in 3% H₂O₂ for 15 min. The sections were then incubated with primary antibodies against LC3B (Abmart, Shanghai, China), Beclin1 (Abmart), p62 (Wanleibio, Shenyang, China), nuclear factor kappa B (NF-κB) p65 (Abmart), or NLRP3 (Wanleibio) at 37°C for 1 h and subsequently incubated with secondary antibodies (PV-9001, ZSGB-BIO, Beijing, China) according to the manufacturer's instructions. The expression levels of related proteins were scored semiquantitatively in a blind manner as the product of intensity scores (0, negative; 1, weak; 2, moderate; and 3, strong) and diffusion scores (1, 0-25% positive cells; 2, 26-50% positive cells; 3, 51-75% positive cells; and 4, >75% positive cells) [19 (link)].

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.

Min6 cells were plated in six-well dishes with overnight culture and then incubated with 10 μmol/L tested compounds and 300 μmol/L PA for 12 h. Total proteins of cells were lysed with RIPA lysis buffer (Boster, China) and the protein lysis was separated by SDS-PAGE and transferred from gel to PVDF membrane (Millipore, USA). Then, the membranes were blocked, stained overnight with corresponding primary antibodies and then with second antibody for 1 h. The signals of detected proteins were measured by Chemidoc Imaging System (BIO-RAD, USA). The antibody used were as follows: TXNIP (Abcam, ab188865), NLRP3 (Wanlei, Wuhan, China; WL02635), Cleaved caspase 1 (Wanlei, Wuhan, China; WL03450), Cleaved caspase 3 (CST, #9664s), GAPDH (Bioworld, Nanjing, China; AP0063).