D3a3z

Differentiated THP-1 cells seeded in 12-well plates (1.0 × 10⁶ cells/well) were primed with 100 ng/mL LPS for 4 h, followed by treatment with the peptide solution or nigericin for 3 h. Culture supernatants were collected and concentrated using AMICON Ultra-10 filters (Millipore). After removal of the supernatant, the cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich). The specimens were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. Immunoblotting was essentially performed as described previously [13 (link)]. The membranes were incubated with primary antibodies specific for IL-1β (D3U3E, Cell Signaling Technology), cleaved IL-1β (D3A3Z, Cell Signaling Technology), or ɑ/β-tubulin (Cell Signaling Technology). After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized using the ECL Advance western blotting substrate (Thermo Fisher Scientific) and a Light-Capture II imaging analyzer (ATTO).

Standard techniques were used for protein quantification, separation, transfer, and blotting in KLE and HEC-1A cells. Primary antibodies immunoblotting antibodies were used: ERRα (1: 500, ab76228, Abcam, London, UK), ERRα (1:500, E1G1J, Cell Signaling Technology, Massachusetts, USA), NLRP3 (1:500; 19,771-1AP, Proteintech, Wuhan, China), HIF-1α (1:500; 20,960-1AP, Proteintech, Wuhan, China), cleaved GSDMD (1:500; #36,425, Cell Signaling Technology, Massachusetts, USA), GSDMD (1:500, E9S1X, Cell Signaling Technology, Massachusetts, USA), GSDME (1:500, 84005S, Cell Signaling Technology, Massachusetts, USA), Caspase1 (22,915–1-AP, Proteintech, Wuhan, China), Caspase-1 (YT5743, Immunoway, USA), Caspase-1 (1:500, D7F10, Cell Signaling Technology, Massachusetts, USA), Caspase-3 (1:500, D3R6Y, Cell Signaling Technology, Massachusetts, USA), IL-18 (1:500,10,663–1-AP, Proteintech, Wuhan, China), cleaved-IL-1β (1:500, D3A3Z, Cell Signaling Technology, Massachusetts, USA), β-actin (1:2000, YM3028l, Immunoway, USA), β-tublin (1:2000, YM3030, Immunoway, USA), and GAPDH (1:2000; YM3029, Immunoway, USA).