Gsdmd 20770 1 ap

GSDMD (20770-1-AP) was purchased from Proteintech, China. Dulbecco's modified Eagle's medium/F12 medium, fetal bovine serum, and trypsin were purchased from Gibco, USA. Antibodies to NLRP3 (ab214185), Caspase-1 p20 (ab1872), GSDMD-N (ab215203), IL-1β (ab200478), IL-18 (ab207324), and GAPDH (ab9485) were purchased from Abcam, UK. Goat anti-rabbit (G1213-100UL) secondary antibody and diaminobenzidine chromogenic reagent (G1212-200T) were purchased from Servicebio, China.

Lysed samples collected by Radio-Immunoprecipitation Assay buffer (Beyotime) were measured by a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). The proteins were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane which was then covered with 5% skim milk for overnight incubation with anti-pro-caspase-1 (ab179515, Abcam), gasdermin D (GSDMD; 20770-1-AP, Proteintech), plasma-membrane-bound pannexin-1 (PANX1; 12595-1-AP, Proteintech), p-p65 (3033, Cell Signaling Technology), p65 (ab86299, Abcam), eIF5A (ab32443, Abcam), and glyceraldehyde-3phosphate dehydrogenase (ABclonal, A19056, 1:1000). Next, horseradish peroxidase-conjugated secondary antibody was added for 1.5 h and enhanced chemiluminescence-developed bands were imaged using Tanon 5200 (Tanon, China) and analyzed using ImageJ software.