Nlrc4

Antibodies targeting the following proteins were purchased from ABclonal Biotech Co., Ltd. (USA): HIF-1α (#A11945), HIF-1α (#A1544), NLRP3 (#A6345), NLRP12 (#A6671), NLRC4 (#A7382), CASP1 (#A0964), CASP8 (#A0215), IL-1β (#A1112) and GSDMD (#A10164). Anti-cleaved-CASP8 (#8592S) and anti-phospho-NF-kB P65 (#3033S) antibodies were obtained from Cell Signaling Technology. An IL-1β neutralizing antibody (#14–7012-85) was obtained from eBioscience. The CASP1 inhibitor (CASP1 inh) Z-YVAD-FMK (YVAD, #ab141388) was obtained from Abcam. The NF-kB P65 inhibitor JSH-23 (#S7351) was purchased from Selleckchem. Anti-β-actin (#AP0060) and secondary antibodies were purchased from Bioworld Technology. Lipofectamine 3000 was purchased from Invitrogen.

Fixed samples were incubated with PBS containing 0.5% Triton X-100 for 20 min. PBS was added and washed three times, followed by blocking with 5% BSA for 30 min. Sections were then washed, and primary antibodies (AIM2, A3356, 1:100, Abclonal, CN; Caspase-1, 22915-1-AP, 1:100, Proteintch, CN; NLRC4, A7382, 1: 100, Abclonal, CN) were added and then incubated overnight at 4°C. A secondary antibody (Cy3 GoatAntiRabbit IgG (H+L) (AS007, 1:200, Abclonal, CN) or FITC Goat AntiRabbit IgG (H+L) (AS011, 1:200, Abclonal, CN)) was added and incubated for 50 min at room temperature (23°C ± 2°C), and cell nuclei were stained with DAPI.