Bs 14287r

Protein extraction was performed using RIPA lysis buffer (Pierce, IL, USA) containing protease inhibitor (Roche, CA, USA). Protein extracts were subjected to 10% SDS-polyacrylamide gel electrophoresis followed by electro-transfer to polyvinylidene difluoride membrane. After 1 h of pre-membrane blocking with 5% BSA, the proteins were incubated with respective primary antibodies at 4 °C overnight followed by secondary antibodies incubation at room temperature for 1 h. The detection of proteins was carried out using ECL reagent. Primary antibodies used in this study include: NRLP3 (1:1000, cat. no. ab214185, Abcam), METTL14 (1:1000, ab223090, Abcam), cleaved caspase-1 (1:1000, ab1872, Abcam), IL-1β (1:1000, ab2105, Abcam) and IL-18 (1:1000, ab18672, Abcam), GSDMD-N (1:1000, bs-14287R, Bioss, Beijing, China) and GAPDH (Invitrogen, cat. no. PA1-987).

Proteins from kidney tissues or cultured TCMK-1 cells were extracted using a Nuclear-Cytosol Extraction Kit (KGP150, Nanjing KeyGen Biotech. Co., Ltd.). Total protein levels were quantified with a BCA assay kit (Invitrogen). The expressions of Tisp40, GSDMD-N, NLRP3, Caspase-1, p65,p-p65 and β-actin was measured. The membranes were incubated overnight with the following antibodies: anti-Tisp40 (1: 100; sc-54800; Santa), anti-GSDMD-N (1:100,bs-14287R, Bioss), anti-NLRP3 (1:200; cat. no. ab214185, Abcam), anti-Caspase-1 (1:200; cat. no. ab138483, Abcam), anti-p65 (1:200; cat. no. ab32536, Abcam), anti-p-p65 (1:200; cat. no. ab86299, Abcam) and β-actin (1:500; cat. no. ab8224, Abcam). Subsequently membranes were incubated with HRP-conjugated secondary antibodies (1:500; cat. no. ab6789, Abcam). Protein bands were visualized and detected using Odyssey Infrared Imaging system Model 9120 (LI-COR Biotechnology).

Heart myocardial tissues were isolated and fixed with 4% Paraformaldehyde followed with incubation with primary antibodies against respective proteins: NRLP3 (1:100, cat. no. ab214185, Abcam, Cambridge, MA), METTL14 (1:100, ab223090, Abcam), cleaved caspase-1 (1:100, ab1872, Abcam) and GSDMD-N (1:100, bs-14287R, Bioss, Beijing, China), to detect their expression levels. Images were visualized using a ZEISS Axio Observer A1 (Oberkochen, Germany) microscope system and processed with ZEISS software.