Ab238124

Western blotting was performed using the standard procedures as we previously reported^{58 (link),59 (link)}. Proteins were extracted from cells or tissues using RIPA lysis buffer (Solarbio). Equal volumes of lysates were loaded and separated on 10% SDS-PAGE gels and blotted on polyvinylidene difluoride membrane. After being blocked with 5% skim milk for 2 h at room temperature, the membranes were incubated with primary antibodies overnight at 4 ˚C. The primary antibody used in western blot analysis included Anti-ASC (1:1000; ab155970; Abcam); anti-caspase-1 (1:1000; ab207802; Abcam); anti-caspase-4 (1:1000; ab238124; Abcam); anti-GSDMD (1:1000; 66387-1-Ig; Proteintech); anti-PD1 (1:1000; ab52587; Abcam) and anti-GAPDH (1:500; ab8245; Abcam;). Anti-rabbit-HRP (1:5000; #7074, Cell Signaling Technology) and anti-mouse-HRP (1:5000; #7076, Cell Signaling Technology) were used as secondary antibodies and incubated at room temperature for 1 h. The blots were detected using a chemiluminescence kit (cat. no. 34577; Thermo Fisher Scientific, Inc.) and imaged using MiniChemi 610 system (Sage Creation Science, Co., Ltd.).

Protein expressions of caspase-1, Capase-4, and cleaved N-terminal gasdermin D (GSDMD-N) were measured by Western blotting [26 (link)]. Proteins were extracted from cells and tissues by RIPA (Sigma-Aldrich, Missouri, USA) reagents and were detected using the BCA method (P0012; Beyotime, Shanghai, China). Next, 30 μg total protein from each well was separated by 10% SDS-PAGE electrophoresis (P0690; Beyotime, Shanghai, China) and transferred to a PVDF membrane (FFP32; Beyotime, Shanghai, China). The membranes were blocked with 5% defatted milk for 1 h. Then, primary antibodies, including anti-caspase-1 (ab207802, 1/1000), anti-Capase-4 (ab238124, 1/1000), and anti-GSDMD-N (ab215203, 1/1000), were added (Abcam, California, USA). The next day, HRP labeled secondary antibody (ab7090, Abcam, California, USA) was added after the membrane was washed with PBS, and the membrane was incubated at room temperature for 1 h. The membranes were developed using ECL reagents with GAPDH as control.

Radioimmunoprecipitation assay (RIPA; Beyotime, China) buffer was applied for protein extraction from tissues. The supernatants were run on 8%–12% acrylamide gels via SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Millipore, USA). Antibodies against GPX4 (#P36969; RayBiotech, USA; 1:1000), NLRP7 (#AB117732; Abcam, USA; 1:1000), CASP6 (#AB108335; Abcam; 1:1000), NLRP6 (#P59044; RayBiotech; 1:1000), IL-6 (#AB271042; Abcam; 1:2000), CASP5 (#AB40887; Abcam; 1:3000), CASP4 (#AB238124; Abcam; 1:1500), TIRAP (#P58753; RayBiotech; 1:1000), and GAPDH (#60004-1-Ig; Proteintech, China; 1:20000) and HRP-conjugated secondary antibodies (#ab97080 or ab47827; Abcam; 1:2000) were employed for western blot. The chemiluminescence western blot detection system (Bio-Rad, USA) was utilized for protein detection.