Ab223090

Cell lysates were prepared with the Radioimmunoprecipitation assay (RIPA) lysis buffer. Antibodies, including anti-METTL14 (ab252562; Abcam, Cambridge, MA, United States), anti-DGCR8 (ab90579; Abcam) or normal IgG antibody (sc-2027; Santa Cruz Biotechnology, Inc.), were incubated with the cell lysates followed by incubation with Protein A/G PLUS-Agarose beads (sc-2003; Santa Cruz Biotechnology, Inc.) for 2 h at 4°C. After washing with the lysis buffer 3 times, the samples were subjected to Western blot analysis using anti-METTL14 (ab223090; Abcam) and anti-DGCR8 (ab191875; Abcam) antibodies.

The cell lysates were prepared using the radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China). The quantitation of protein in the samples was performed using the bicinchoninic acid (BCA) Protein Assay Kit (Beyotime). For SDS-PAGE, 20 μg of protein sample was loaded to each well followed by the transmembrane onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States). The blocking of the membrane was conducted by incubating with 5% fat-free milk and then primary antibodies against METTL3 (ab195352; Abcam), METTL14 (ab223090; Abcam), WTAP (ab195380; Abcam), SIRT1 (ab110304; Abcam), and GAPDH (#5174, Cell Signaling Technology) overnight at 4°C. The blots were then washed in Tris-buffered Saline Tween-20 (TBST) three times and then incubated with secondary antibodies (A0208, A0216; Beyotime, Shanghai, China). The blots were examined by chemiluminescence using the Enhanced Chemiluminescence Detection kit (Pierce Biotechnology, Rockford, IL, United States). After exposure, the intensity of bands was analyzed by Image-Pro Plus 6.0 software.

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).