Ab219413

Cells were rinsed in PBS and scraped in 400 ml of cell lysis buffer containing protease and phosphatase inhibitor cocktails (Roche, Mannheim, Germany). Cell lysates were then centrifuged at 12,000 rpm for 15 min at 4°C. The protein concentration in each lysate was then determined using the BCA assay. SDS-PAGE gels were loaded with the same amount of protein, and the NC membrane was blocked for 30 min with skimmed milk. Several primary antibodies were used for western blotting (as 1:1000 dilutions): NF-κB p65, ab16502; IkBα, ab76429; TLR2, ab108998; MyD88, ab219413; IRAK1, ab238; TRAF6, ab33915; TIRAP, ab17218; TAK1, ab109526; p-NF-κB p65, ab76302; IRAK4, 4363; p-PI3K, ab182651 (Abcam, Cambridge, MA, United States). We also used β-actin, 4970, GAPDH, 5174; AKT, 9272; and p-AKT, 4060S (Cell Signaling Technology, United States). HRP-labeled goat anti-rabbit secondary antibodies were used at a dilution of 1:2000 (A0208; Beyotime Biotechnology, Shanghai, China) and immunoreactive bands were detected using an Amersham Imager 600 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

Cell or tissue proteins were extracted using RIPA solution (P0013C, Beyotime, Shanghai, China) containing protease inhibitors (CW2200S, CWBIO, Beijing, China). After extraction and denaturation of total proteins, 10% gel electrophoresis was used to separate the proteins, and activated PVDF membranes were used for transfer. Subsequently, the blocked-membranes with 5% skim milk powder were incubated with primary antibodies such like anti-TLR4 (1:1000, 14358S, CST, Boston, MA, USA), anti-MyD88 (1:1000, ab219413, Abcam, Cambridge, UK), anti- TIR domain containing adaptor molecule 1 (TRIF) (1:3000, ab13810, Abcam, Cambridge, UK), anti-NF-κB (1:3000, 8242T, CST, Danvers, MA, USA), anti-IκBα (1:3000, 4814T, CST, Danvers, MA, USA), Anti-ATP citrate lyase antibody (ACLY) (1:10000, ab40793, Abcam, Cambridge, UK), phospho- ACLY (1:1000, 4331T, CST, Danvers, MA, USA), and anti- β-actin (1:20000, 81115-1-RR, Proteintech, Chicago, IL, USA) followed by corresponding secondary antibodies (1:6000, 7074/7076, CST, Danvers, MA, USA) at 25 °C for 2 h. The membrane was incubated with ECL reagents (610020-9Q, Qing Xiang, Shanghai, China) and visualized by ImageJ software.

Total protein was extracted from mice’s heart and colon tissues using radio-immunoprecipitation assay (RIPA) lysis buffer, and protein concentrations of the extracts were assessed by BCA assay. Western blotting was performed using the following antibodies: TLR4 (1:800, Abcam, ab- 13556), MyD88 (1:1000, Abcam, ab-219413), NF-κB p65 (1:10,000, Abcam, ab-32536), NF-κB p65 (phospho-T254) (1;800, Abcam, ab-131100), occludin (1:1000, Abcam, ab-216327), GAPDH (1:1000, Abcam, ab-32536), and β-actin (1: 500, Abcam, ab-115777). Band intensities were quantified using Image Pro Plus 6.0.

Lung samples were homogenized and qualified with a BCA detecting kit (Beyotime, China). Protein lysates of 30 μg were separated by SDS‐PAGE and transferred onto the PVDF membrane (Millipore, USA). After being blocked with 5% (w/v) nonfat milk, the membranes were then incubated with primary antibodies against TLR4 (1:500, ab13556, Abcam), MyD88 (1:1000, ab219413, Abcam), NF‐κB p65 (1:2000, ab32536, Abcam), Bax (1:1000, ab182734, Abcam), Bcl‐2 (1:2000, ab182858, Abcam), and β‐actin (1:5000, D191047, Sangon Biotech, China) at 4°C overnight. Next, the membranes were incubated with HRP‐conjugated goat anti‐rabbit secondary antibody (1:2000, ab7090, Abcam) for 1 h at room temperature. Finally, the optical density of bands was measured via ImageJ software.

Proteins were extracted from the PVN samples by protein lysates, and the protein concentrations were measured using the BSA assay. Equal amounts of protein samples were separated by a 10%–12.5% gel and incubated with TLR4 antibody (GeneTex, GTX64330, 1:1000), anti‐MyD88 antibody (Abcam, ab219413, 1:1000), anti‐NF‐kB p65 (Abcam, ab16502, 1:1000), anti‐IkB alpha antibody (Abcam, ab32518, 1:1000), GAPDH (Cell Signaling, #2118, 1:1000) and anti‐histone antibody (Bios, bs‐17422R, 1:1000) overnight at 4°C. Then, they were incubated with HRP goat anti‐mouse IgG (H + L) (ABclonal, AS003, 1:2000) and goat anti‐rabbit IgG‐HRP (Absin Bioscience Inc., abs20040ss, 1:5000) for 1 h at room temperature on a shaker. The protein strips were developed with Stain‐Free Western blotting technology (Bio‐Rad) and analysed with ImageJ software (Bio‐Rad, v.1.8.0.112).

The following antibodies were used for immunoblotting, immunofluorescence, and flow cytometry analysis: anti-Caspase-1 (ab179515; Abcam), anti-NLRP3 (ab263899; Abcam), anti-ASC (sc514414; Santa Cruz), anti-NF-κB P50 (ab32360; Abcam), anti-NF-κB P65 (cst6956; Cell Signaling Technology), anti-phospho–NF-κB P65 (cst3033; Cell Signaling Technology), anti-MyD88 (ab219413; Abcam), anti-IKK-β (ab124951; Abcam), anti-TRAF6 (ab33915; Abcam), anti-GPR81 (ab106942; Abcam), anti-tubulin (11224; Proteintech), anti-pro-IL-1β (cst12242; Cell Signaling Technology), anti–F4/80-PE (123110; BioLegend), anti–CD11b-BV421 (101236; BioLegend), anti–CD68-APC (137007; BioLegend), anti–CD206-AF647 (141712; BioLegend), anti–Ly6G-FITC (11–9668-80; eBioscience), anti-CD16/32 (14–0161-82; eBioscience), anti-CD11b (2185-1-ap; Proteintech), anti-F4/80 (cst70076; Cell Signaling Technology), anti-Ly6G monoclonal antibody (65140-1-ig; Proteintech).
The following reagents were used for this study: caerulein (C9026-1 MG; Sigma, Munich, Germany), ELISA kit for IL-1β, IL-6 and TNF-α (Lianke), alpha-amylase determination kit (BIOSINO, Beijing, China), PBMC isolation kit (LTS1092; TBD), LPS from Escherichia coli O55:B5 (Sigma, Germany), L-lactate assay kit (ab65330; Abcam), sodium L-lactate (L7022; Sigma), L-lactic acid (L1750; Sigma).