Ecl substrate

Cells were collected and lysed. Total protein was extracted and quantified using the BCA Protein Assay Kit (Solarbio), following the manufacturer’s instructions. Primary antibodies (all Abcam) used included anti-NLRP3 (1:1000), anti-cleaved caspase-1 (1:1000), anti-IL-1β (1:1000), anti-E-cadherin (1:1000), anti-vimentin (1:1000), and anti-β-actin (1:5000) and were incubated at 4 °C overnight. After washing with tris-buffered saline with Tween (TBS-T) three times, the secondary antibody (Solarbio) was added for 1 h at room temperature. The ECL substrate (Solarbio) was used to detect the expression of target proteins in the FUSION FX5 imaging system (Bio-Rad, Hercules, CA, USA).

The A2780 and A2780DDP were purchased from MEIXUAN biological science and technology (China, Shanghai). The cells were mycoplasma-free and authenticated by STR analysis. We strictly followed the instructions of the lysate kit to extract the protein, and the protein was separated on polyacrylamide gels under appropriate conditions and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membrane was subsequently blocked for 2 h using 5% skimmed milk and incubate the primary antibody GAPDH (1: 100000; Proteintech: 60004-1-Ig) at 4°C overnight. The next day, the diluted secondary antibody HRP-conjugated goat anti-rabbit IgG (1: 10000; Proteintech: SA00001-2) was incubated at room temperature for about 1 hour. Membranes were incubated with electrochemical luminescence (ECL) substrate (Solarbio, China) and then exposed to an X-ray film.

TLR4 is overexpressed in colonic mucosal DCs of patients with IBD [13 (link)]. So the protein samples of colonic mucosa were prepared as described for ELISA. The protein concentration was determined by a BCA protein assay kit (CoWin Biotech, Jiangsu, China). Equal weight of protein per sample was separated using 10% SDS-PAGE (CoWin Biotech, Jiangsu, China) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). These membranes were blocked at room temperature for 1 h and then incubated overnight with the following primary antibody at 4°C, including anti-GAPDH (No. ab181602) (1 : 2000), TLR4 (No. ab13556) (1 : 500), MyD88 (No. ab2064) (1 : 1000), TRAF6 (No. ab40675) (1 : 5000), TAB2 (No. 3745S) (1 : 1000), IκB (No. 4814S) (1 : 1000), and NF-κBp65 (No. ab16502) (1 : 1000). All antibodies were purchased from Abcam (Cambridge, UK) except IκB and TAB2 from Cell Signaling Technology (Boston, USA). Next, samples were incubated with secondary antibodies for 1 h at room temperature. The immunoreactive bands were assessed using the ECL substrate (Solarbio, Beijing, China). The images were captured using the Highly Sensitive Chemiluminescence Imaging system (UVP ChemStudio 515, Analytik Jena, Germany). The images were analyzed using Image-Pro Plus 6.0 (Media Cybernetic, USA).

Primary antibodies against AKT (#4685S), p-AKT (#4060T), β-catenin (#8480T), CD63 (#55051S), CD81 (#56039) and CD9 (#13403) were purchased from Cell Signaling Technology, USA. The HRP-labeled anti-rabbit IgG monoclonal antibody (#D110065, Sangon Biotech, China) was used as the secondary antibody, and the fluorescent signal was developed with ECL substrate (#SW2010, Solarbio, China). The chemiluminescent signals were captured using a Bio-Rad ChemiDoc XRS system (Bio-Rad, USA).

Total proteins were extracted from mouse heart tissues and H9C2 cells using RIPA lysis buffer. Proteins from the serum of participants were extracted using the serum protein extraction kit (Solarbio, Beijing, China). After concentration was measured using a BCA protein assay kit (Solarbio), the protein samples were run on 15% SDS-PAGE and transferred to polyvinylidene fluoride membranes. After blocking, the membranes were incubated with primary antibodies targeting IL-33 (ab118503; Abcam, Cambridge, UK), sST2 (ab25877; Abcam), TCF7L2 (sc-271287, Santa Cruz Biotechnology, Santa Cruz, CA, USA), GAPDH (ab9485; Abcam) at 4°C overnight, followed by incubation with anti-rabbit (ab6721; Abcam) or anti-mouse (ab205719; Abcam) conjugated to horse radish peroxidase at 25°C for 1 h. The band signals were visualized using an ECL substrate (Solarbio).

Total proteins were harvested from the cortex and hippocampus. The proteins were transferred to polyvinylidene fluoride membranes (Millipore) that were blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature and incubated overnight with the following primary antibodies at 4℃: anti‐p‐RIP3 (Abcam), anti‐p‐MLKL (Abcam), anti‐tumour necrosis factor‐α (TNF‐α) (Abcam), anti‐IL‐1β (Abcam), anti‐IL‐6 (Abcam) and anti‐β‐actin (Cell Signaling Technology). After incubation with secondary antibodies (Solarbio), the positive bands were visualized using an ECL substrate (Solarbio). For immunohistochemical analysis, brain tissue sections were incubated overnight with the following primary antibodies at 4℃: anti‐p‐RIP3 (Abcam) and anti‐p‐MLKL (Abcam). Subsequently, a PV9000 kit (ZSGB‐BIO) was used for follow‐up. For immunofluorescence analysis, brain tissues were blocked with goat serum for 1 hour and incubated with primary antibodies against p‐RIP3 (Abcam), p‐MLKL (Abcam), NeuN (Abcam), Iba‐1 (Wako; Servicebio) and GFAP (Servicebio) overnight at 4℃. Then, the tissues were incubated with Alexa Fluor® 594 goat anti‐rabbit IgG and Alexa Fluor® 488 goat anti‐mouse IgG secondary antibodies (Thermo Fisher Scientific). Nuclei were counterstained with DAPI (Thermo Fisher Scientific).