Anti tlr4

Occludin and ZO-1 are vital tight junction proteins to maintain barrier function (25 (link)). Toll-like receptor 4 (TLR4) and its downstream myeloid differentiation factor 88 (MyD88) form an important pathway to promote the activation of microglia (26 (link)). The relative levels of Occludin and ZO-1 expression in the intestine and brain and TLR4 and MyD88 expression in the brain to the β-actin were quantified by Western blotting. Briefly, each type of tissue samples were homogenized in lysis buffer (Servicebio) and centrifuged. After determining protein concentrations using the bicinchoninic acid method, the lysates (30 μg/lane) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 10% gels and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat dry milk in phosphate-buffered saline (PBS) containing Tween 20 and probed with anti-Occludin, anti-ZO-1, anti-TLR4, and anti-MyD88 (Cell Signal Technology) at 4°C overnight. After being washed, the bound antibodies were detected with appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Servicebio) and visualized with enhanced chemiluminescence.

Dulbecco Modified Eagle Medium (DMEM) was obtained from GIBCO (USA). Fetal bovine serum (FBS) was obtained from Biolnd (Aus). The total RNA extraction kit (TRIzol) was purchased from TaKaRa (Jap). Trans Script assay kit was obtained from Beijing Trans Gen Biotech Co. Ltd. (CN) and qPCR assay kit was from Invitrogen (USA). ICAM-1, VCAM-1, IL-6 gene primers were obtained from Life Technologies (USA). Anti-ICAM-1, anti-p38, anti-p-p38, anti-SAPK/JNK, anti-p-SAPK/JNK, anti-ERK 1/2, anti-p-ERK1/2, anti-TLR4 and anti-SCD-1 were purchased from Cell Signaling Technology (USA). TAK242 was purchased from Med Chem Express (USA). Recombinant human leptin was purchased from Peprotech (USA). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphe-nyltetrazolium (MTT), elaidic acid (9t18:1) and vaccenic acid (11t18:1) were purchased from Sigma (St. Louis, USA) with purity over 99%. The CLA isomer (9-cis, 11-trans-CLA) was purchased from Cayman Chemical Company (AnnArbor, MI, USA) with purity over 96%. 9t18:1, 11t18:1 and 9c11t-CLA were dissolved in 0.1 mM sodium hydroxide solution at 65 °C.

DEAE Sepharose fast flow, Phenyl Sepharose CL-4B and Sephadex G-75 were purchased from GE Healthcare (Shanghai, China). Tris and sodium dihydrogen phosphate, dimethylsulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma Chemical Co. (St Louis, MO, United States). NO Griess reagent kit was obtained from Beyotime Corp (Shanghai, China). FITC anti-mouse CD86, FITC-dextran, and PE anti-mouse MHC II were obtained from Biolegend Corp (Shenzhen, Guangdong, China). DMEM medium, RPMI-1640 medium and fetal bovine serum (FBS) were purchased from Gibco Invitrogen Corp (San Diego, CA, USA). Murine TNF-α and IL-6 ELISA test kits were obtained from Excell Corp (Shanghai, China). ECL Western blotting detection kit was obtained from Tanon Crop (Shanghai, China). Anti-TLR2, anti-TLR4, anti-p-STAT3, anti-STAT3, anti-MAPKs, anti-p-MAPKs, anti-Akt, and anti-p-Akt primary antibodies were provided by Cell Signaling Technology (Beverly, MA, USA). All other reagents used in the experiments were of analytical grade.
RAW264.7 cell line and YAC-1 cell line were obtained from Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and were maintained in DMEM supplemented with 100 IU mL⁻¹ penicillin, 100 μg/mL streptomycin and 10% FBS at 37 °C under humidified air with 5% CO₂.

Frozen colon samples and NCM-460 cells from each group were homogenized in a 5× volume of homogenization buffer (Tris HCl, 50 mM; pH, 7.4; NaCl, 100 mM; EDTA, 10 mM; and Triton X-100, 0.5%) with protease inhibitors (Roche, Mannheim, Germany) at 4 °C. The homogenates were centrifuged at 10,000 rpm for 5 min at 4 °C, and the supernatants were collected. Total protein concentration was measured by the Bradford protein assay (Bio-Rad, Hercules, CA). Proteins were lysed with 1× SDS loading buffer, and the lysates were boiled at 100 °C for 5 min and centrifuged at 10,000 rpm for 1 min. Approximately 40 μg of total protein was loaded onto an SDS-PAGE gel and resolved at 120 V for 1–2 h. After that, the proteins were transferred to a PVDF membrane at 300 mA for 2.5 h. The membranes were then washed with 1× TBST 3 times for 10 min each and incubated with secondary antibodies at room temperature for 1 h. Finally, the membranes were incubated with ECL and exposed. The following antibodies from Cell Signaling Technology (USA) were used: anti-HMGB1, anti-TLR4, anti-IL-33, anti-ZO-1 and anti-occludin.

Protein isolation from liver tissue and Western blot analysis were performed as previously described (Dou et al., 2018 (link)). The following antibodies were used: anti-phosphorylated-AMPKα (Thr172), anti-AMPKα, anti-PPARα, anti-CPT-1, anti-acetyl-CoA-carboxylase (ACC), anti-phosphorylated-ACC, anti-SREBP-1c, anti-fatty acid synthase (FAS), anti-DGAT-2, anti-P53, anti-Bcl2, anti-Bax, anti-TLR4, anti-JNK, anti-phosphorylated-JNK, anti-phosphorylated-P38, anti-P38, anti-phosphorylated-ERK, anti-ERK, anti-cleaved-caspase-3, and anti-GAPDH (Cell Signalling Technology, Danvers, MA, United States).

The following primary antibodies were used: mouse monoclonal anti-β-Actin (12262, 1:1000, Cell Signaling), mouse monoclonal anti-GAPDH (51332, 1:1000, Cell Signaling), rabbit polyclonal anti-BIG1 (A300-998A, 1:1000, Bethyl), rabbit monoclonal anti-IκBα (4814, 1:1000, Cell Signaling), rabbit monoclonal anti-pS536-p65 (3033, 1:1000, Cell Signaling), rabbit monoclonal anti-pS176/180-IKKα/β (2697, 1:1000, Cell Signaling), rabbit monoclonal anti-TLR4 (14358, 1:1000, Cell Signaling), rabbit monoclonal anti-MyD88 (4283, 1:1000, Cell Signaling), rabbit polyclonal anti-TIRAP (ab17218, 1:1000, Abcam), mouse monoclonal anti-PIP2 (sc53412, 1:200, Santa Cruz), rabbit polyclonal anti-ARF1 (PA1-127, 1:2000, Thermo Fisher), mouse monoclonal anti-ARF3 (610784, 1:500, BD Biosciences), mouse monoclonal anti-ARF5 (H00000381-M01, 1:500, Abnova), rabbit polyclonal anti-ARF6 (ab77581, 1:1000, Abcam), and mouse monoclonal anti-Myc-Tag (2276, 1:1000, Cell Signaling). Chemical reagents used are as follows: LPS (L4516, Sigma-Aldrich; L23351, Invitrogen), R848 (tlrl-r848-5, Invitrogen), CpG ODN (tlrl-2395-5, Invitrogen), FSL-1 (tlrl-fsl, Invitrogen), Pam3csk4 (tlrl-pms, Invitrogen).

The levels of Occludin and ZO-1 in the intestine and TLR4 and MyD88 in the brain relative to β-actin were quantified by Western blotting. In brief, tissue samples were homogenized in lysis buffer (Servicebio, Wuhan, China) and centrifuged. After the protein concentrations were determined using the bicinchoninic acid method, the lysates (30 μg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% gels and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS) containing Tween 20 and probed with anti-occludin, anti-ZO-1, anti-TLR4 and anti-MyD88 (Cell Signaling Technology, Denver, United States) at 4°C overnight. After being washed, the bound antibodies were detected with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Servicebio, Wuhan, China) and visualized with enhanced chemiluminescence.