Anti caspase 1 antibody

Cells were washed twice with ice-cold phosphate buffered saline (PBS) and then harvested and lysed in cold radioimmunoprecipitation assay (RIPA) buffer. Equal amounts of protein were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). The membrane was blocked with 5% nonfat milk and incubated with anti-NLRP3 antibody, ASC antibody, anti-caspase-1 antibody, and anti-IL-1β antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Blots were developed using enhanced chemiluminescent substrate (Thermo Fischer Scientific Pierce, IL, USA).

To evaluate the macrophage-platelet interaction during DBV infection, 8 × 10⁵ PMA-activated THP-1 cells were seeded onto a confocal plate (Biosharp, Anhui, China) and infected by DBV (MOI = 1). Macrophages treated with LPS and nigericin or untreated were served as positive and negative controls, respectively. After 72 h incubation, macrophages were washed three times with precooled PBS, fixed with 1% paraformaldehyde for 5 min, and penetrated with 0.1% Triton-X for 10 min. After blocking with immunostaining blocking solution (Beyotime, Shanghai, China) for 10 min, samples were incubated overnight at 4°C with anti-tubulin antibody (1:200) (Abcam, Britain), anti-caspase-1 antibody (1:200) (Santa Cruz Biotechnology, USA) and anti-DBV-Gc antibody (1: 200) (Sangon Biotech, Shanghai, China). Samples were washed with PBS and incubated with donkey anti-rabbit IgG H&L (Alexa Fluor 568) pre-adsorbed (1:200) (Abcam, Britain), goat anti-mouse IgG H&L (Alexa Fluor 488) pre-adsorbed (1:200) (Abcam, Britain) and donkey anti-rat IgG H&L (Alexa Fluor 647) pre-adsorbed (1:200) (Abcam, Britain) for 1 h. The nuclei were then stained using the mounting medium with DAPI (Abcam, Britain). The images were obtained using a laser confocal microscope (ZEISS, LSM880, Germany).

Ileum tissues were homogenized, and the resulting total protein was extracted by RIPA lysis mixed with PMSF (Solarbio, Beijing, China). Then, 50 μg of protein per sample was subjected to 7.5%, 10%, or 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Solarbio, Beijing, China). After overnight incubation at 4°C with anti-NLRP3 antibody (dilution at 1 : 1000, Abcam, Cambridge, UK), anti-caspase-1 antibody (dilution at 1 : 300, Santa Cruz, Oregon, USA, SC-398715), anti-IL-1β antibody (dilution at 1 : 600, Bioss, Beijing, China), anti-IL-18 antibody (dilution at 1 : 600, Bioss, Beijing, China), anti-β-actin antibody (dilution at 1 : 2000, Servicebio, Wuhan, China), and anti-HSP-90 antibody (dilution at 1 : 1000, Santa Cruz, Oregon, USA), the membranes with blotted proteins were then incubated with HRP-conjugated goat anti-rabbit secondary antibody (dilution at 1 : 2000, CST, Boston, USA) or rat anti-mouse secondary antibody (dilution at 1 : 2000, Servicebio, Wuhan, China) for an hour at room temperature. After washing three times with TBST, the electrochemiluminescence solution (ECL, Millipore, Massachusetts, USA) was added to the membranes, and then, the membranes were exposed to the exposure machine (ChemiScope series, Clinx Science Instruments Co., Ltd), and the resulting images were recorded and analyzed.

L-NAT was obtained from Sigma Aldrich (St. Louis, MO., USA). Rat ELISA kit was purchased from NeoBioscience (Shanghai, China). The cell counting kit-8 (CCK-8) came from 7sea-Biotech (Shanghai, China). Anti-ASC, NLRP3, IL-1β, TLR4 and NF-κB were purchased from Bioss Antibodies (Beijing, China). Anti-Caspase 1 antibody came from Santa Cruz Biotechnology (Shanghai, China). TRIzol reagent was obtained from Life Technologies (USA). RTPA lysis was purchased from SolarBio Life Sciences (Beijing, China).

Proteins were extracted from colon tissues or HT-29 cells and were lysed and homogenized in a buffer containing 50 mM Tris-Cl at pH 8.0, 150 mM NaCl, 0.1% SDS, 1 mM EDTA, 1% Triton‒X, 0.05% sodium deoxycholate, and protease inhibitors. Protein samples were fractionated on 12% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes for 70 min at 100 volts (Bio-Rad). Blots were incubated with primary anti-Gal-3 antibody (1:2000, sc-20157, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-caspase-1 antibody (1:2000, Santa Cruz), anti-GRP78 antibody (1:2000, ABCAM, Cambridge, MA, USA), anti-XBP-1 antibody (1:2000, NOVUS BIO, Littleton, CO, USA), and anti-IL-1β antibody (1:2000, R&D system, MN, USA) followed by horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence (ECL) reagents using the ECL kit (Thermo Scientific, MA, USA). Anti-β-actin (Santa Cruz Biotechnology) was used as the loading control.