Anti traf6

Whole protein in the serum was collected using a whole-protein extraction kit (Nanjing KeyGEN Biotech, Nanjing, China) according to the manufacturer’s instructions. The protein concentration was measured using a bicinchoninic acid kit (Nanjing KeyGEN Biotech). The different proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked in Tris-buffered saline and Tween 20 (TBST) blocking buffer that contained 5% nonfat dried milk for 2 h at room temperature and immunoblotted with primary specific antibodies (anti-TLR4, anti-TRAF6, anti-TAK1, IκB-α, p-IκB-α, anti-NF-κB (p65), anti-p-NF-κB (p-p65), and anti-β-actin (Abcam, UK). After washing with TBST, the strips were incubated with horse-radish peroxidase-conjugated goat anti-mouse IgG (HuaAn, Hangzhou, China). Proteins were detected using a SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermofisher) and visualized using a ChemiDoc™ MP Imaging System (Bio-Rad).

Cells were cultured or treated by rBFT1, followed by harvesting at predetermined time points. After extensive washing, cells were lysed using Whole Cell Lysis Assay (KeyGEN Biotech, China) according to the manufacturer’s instructions and proteins from the lyste were harvested by centrifugation at 12,000 × g for 5 min at 4°C. Protein concentrations were determined by the BCA Assay kit (KeyGEN Biotech, China). Total protein (42 kDa) from each lysate was then separated by SDS-PAGE and subsequently transferred onto PVDF membranes (Millipore, MA, USA). Afterwards, the membranes were blocked with 5% non-fat milk in TBST at room temperature for 1 h, followed by incubation with antibodies against protein of interest overnight at 4°C. The antibodies used in this study included anti-NF-κB/P65 (1:100 in PBS), anti-Macrophage Inflammatory Protein 1α/CCL3 (100 μg/mL in PBS), anti-CCR5 (1:100 in PBS), anti-PI 3 Kinase/P85α (1:200 in PBS), and anti-TRAF6 (1 µg/mL in PBS) (Abcam, CA, USA). After washing with TBST, membranes were striped with appropriate HRP-conjugated secondary antibodies (1:5,000, Abcam, CA, USA) for 1 h at room temperature, followed by visualization using the enhanced Western Bright ECL reagents (Cell Signaling Technology, USA). Protein bands were scanned and analyzed by a chemiluminescence detection system (Bio-Rad, USA).

Cells were collected and lysed in M-PER Mammalian Protein Extraction Reagent. All samples were normalized by their protein concentrations and separated in 10% SDS-PAGE gels and then transferred to membranes (Washington, NY) using the wet transfer blotting system (Hercules, CA). The antibodies used for Western blotting were: anti-CD63 (Abcam), anti-TRAF6 (Abcam), anti-TLR4 (Abcam), anti-NF-kB (Abcam), anti-IRKA1 (Abcam), and anti-GAPDH (Abcam), at a dilution of 1:1000. Then, protein bands were developed using ECL reagents, whose images were acquired using the ChemiDoc Imaging system. This western blot examination was repeated three times.

Trizol reagent, RNAios and Real-time PCR kit were obtained from TaKaRa Company (Dalian, China). Anti- MyD88, anti-TRAF6, anti-TRIF and anti-TLR4 antibodies were offered from Abcam Biotechnology (MA, USA). LPS was taken from Sigma-Aldrich (MO, USA). The immunohistochemistry kits and HRP-labeled secondary antibodies were bought from Boster Biotechnology (Wuhan, China). The IL-1β and IL-6 ELISA kit were purchased from Dkewei Biotechnology (Beijing, China). Corilagin was received from China National institutes for food and drug control.

Cells were lysed in laemmli (Sigma, USA). Protein concentration was assessed by bicinchoninic acid (BCA) assay. Total protein lysates were separate by Tricine-SDS-PAGE and transfer onto 0.22-μm nitrocellulose (NC) filter membranes (Millipore, Bedford, MA, USA). Antibodies used were anti TRAF6 (Abcam, MA, USA). β-actin was used as a control. Western blots were quantified by Image J software. All experiments were performed in triplicate, and the representative results were shown.