Anti il 18

Equal amounts of protein samples (28 μg) were subjected to a SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 5% milk in TBST for 2 h followed by incubation with the following primary antibodies as follows: anti-p-NF-κB P65 (1:1000, Cell Signaling Technology), anti-NF-κB P65 (1:1000, Cell Signaling Technology), anti-Iba-1 (1:1000, Abcam), anti-β-actin (1:7500, Bioss antibodies), anti-GR (1:1000, Bioss antibodies), anti-NLRP3 (1:1500), anti-ASC (1:500), anti-pro-caspase 1 (1:500), anti-caspase-1 p20 (1:750), anti-pro-IL-1β (1:10000), anti-IL-1β (1:750), anti-IL-18 (1:1000) and anti-Lamin B (1:750) both from Wanlei Biotechnology (Shenyang, China). After three times washing with TBST, the membranes were incubated with appropriate secondary antibody. The protein bands were visualized by the ECL detection system (Thermo Scientific, Waltham, MA, United States) and quantified using Image J software. Nuclear and Cytoplasmic Protein Extraction Kit (Applygen Technologies, Co., Ltd., Beijing, China) was used to extract the cytoplasmic/nuclear proteins of NF-κB P65 according to the manufacturer’s protocol.

RIPA lysate (Thermo Fisher Scientific, MA, USA) was applied to colon tissues and cells and protein concentration was measured by the BCA method. Protein samples (200 μg) were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein was transferred to a PVDF membrane (Millipore, Billerica, MA, USA) and blocked with 5% skim milk to reduce non-specific antigens exposure. The PVDF membranes were incubated with the primary antibodies: anti-CD9 (1:500; Proteintech Group), anti-CD63 (1:500; Abcam, Cambridge, UK), anti-CD81 (1:500; Proteintech Group), anti-HSP70 (1:500; Abcam), anti-Alix (1:500; Cell Signaling Technology), anti-Calnexin (1:500; Abcam), anti-NLRP3 (1:1000; Novus), anti-ASC (1:1000; Novus), anti-Caspase-1 p45 (1:1000; Proteintech Group), anti-Caspase-1 p20 (1:200; Santa Cruz Biotechnology), anti-IL-1β (1:500; Cell Signaling Technology), anti-IL-18 (1:200; Wanleibio, Shenyang, China), anti-GSDMD (1:500; Cell Signaling Technology), and anti-β-actin (1:10000; Abclonal, Boston, MA, USA) at 4 °C overnight, followed by the HRP-conjugated secondary antibodies for 30 min at 37 °C. A chemical gel imaging system (GE Healthcare Life sciences China, Beijing, China) was used to visualize protein bands and generate images.