Anti cleaved caspase 1

Proteins were separated from cells or tissues using RIPA buffer (EMD Millipore, Billerica, MA, USA). Then, protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher). The same concentration of samples was separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Bio–Rad Laboratories Inc., Hercules, CA, USA). Next, membranes were blocked with 5% non-fat dry milk at room temperature for 2 h. Then, they were incubated at 4℃ for 24 h with the following primary antibodies: anti-NLRP3 (1:500, Abcam, Cambridge, MA, USA), anti-pro-caspase-1 (1:1,000, Abcam), anti-cleaved-caspase-1 (1:500, Adipogen, San Diego, California, USA), anti-GSDMD-FL (1:500, Abcam), anti-GSDMD-N (1:500, Abcam), anti-Ngb (1:500, Abcam), and anti-GAPDH (1:1,000, Abcam). At room temperature, the membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h. An enhanced chemiluminescence (ECL) kit (Bio–Rad, USA) was used to image the protein bands.