Nbp2 33422

Human renal TECs and epithelial cell medium were obtained from ScienCell. Iohexol was obtained from GE Healthcare and Mannitol was obtained from Sigma. Mouse anti-rabbit IgG and Mouse anti-rat IgG antibodies were purchased from Cell Signaling Technology. We obtained antibodies targeting caspase-11 (Sigma, clone 17D9), caspase-4 (Abcam., ab40887), caspase-5 (Abcam, ab25898), IL-1β (R&D Systems, AF-401-NA), GAPDH (Cell Signaling Technology, #2118), Gsdmd (Novus Biologicals, NBP2-33422), and cytokeratin-18 (Abcam, ab668). Pan-caspase inhibitor Z-VAD-FMK was obtained from R&D Systems.

Total proteins were extracted from mice corneas or HCECs using a cell lysis buffer (Solabao, #R0010) and measured by the BCA protein assay kit (Thermo Scientific, #23227). Equivalent protein samples were separated by 4-20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, USA), and then transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% skimmed milk diluted in TBST for 1 h and then incubated with primary antibodies at 4°C overnight. Primary antibodies targeting NLRP3 (1:500, ABcam, #ab263899), CASP1 (1:200, ABclonal, #A0964), ASC (1:200, Santa Cruz Biotechnology, #sc-514414), IL-1β (1:200, CST, #63124 or #83186), IL-18 (1:200, ABclonal, #A16737), GSDMD (1:500, Novus Biologicals, #NBP2-33422), and β-actin (1:1000, ABcam, #ab8226) were used. Then, the membranes were incubated with HRP-conjugated secondary antibodies (1:2000, CST, #7074 or #7076) for 1 h at room temperature. The immunoblots were detected with enhanced chemiluminescence (ECL, Thermo Scientific, #32106) reagents and recorded by Image Lab software (Bio–Rad, USA).

Cells or tissue lysates and immunoblotting analysis were performed as described previously (Zhang Z. et al., 2020 (link)). Densitometric analysis was conducted using ImageJ software. The primary antibodies used for Western blot included caspase-1 (1:1,000, Abcam, United Kingdom, ab1872), GSDMD (1:1,000, Novus Biologicals, United States, NBP2-33422), caspase-4 (1:1,000, Abcam, United Kingdom, ab238124), caspase-11 (1:1,000, Abcam, United Kingdom, ab262899), and GAPDH (1:10,000, Abcam, United Kingdom, ab181603). All the experiments were repeated at least three times.