Proteins were extracted from colon tissues of around 1.5 cm, adjusted using the Bradford method and Western Blotted as previously described15 (link). Specific primary antibodies were: S1PR3 (Item No. 10006373, Cayman Chemical, Ann Arbor, Michigan, USA, 1:1000); TLR4 (sc-293072, Santa Cruz Biotechnology, Dallas, TX, USA, 1:1000); p-p38 (sc-17852R, Santa Cruz Biotechnology, 1:1000); iNOS (ab15323, Abcam, Cambridge, UK, 1:1000); ZO-1 (ab216880, Abcam, 1:1000). The blots were cut prior to hybridization with antibodies due to the limited amount of protein extract available. Blots were imaged using an Odyssey Fc System (Li-COR Biosciences, Lincoln, NE, USA) and quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA). All densitometries were obtained in arbitrary units of optical density and expressed as a percentage of the control group (100%). Several exposition times were analyzed to ensure the linearity of the band intensities. Loading control (blots shown in the respective figures) was β-actin (A5441, MilliporeSigma). All full-length data of western blots are included in Supplementary Information (see Supplementary Figs. S4 , S5 , S6 , S7 , S8 online).
Tlr4 sc 293072
Manufactured by Santa Cruz Biotechnology
Sourced in United States, China
TLR4 (sc-293072) is an antibody product offered by Santa Cruz Biotechnology. TLR4 is a protein that functions as a receptor, recognizing pathogen-associated molecular patterns. This antibody can be used for the detection of TLR4 in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey fc system, by LI COR (1 mentions)
Ab216880, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Ab15323, by Abcam (1 mentions)
Scanning electron microscope, by Leica (1 mentions)
Ab125212, by Thermo Fisher Scientific (1 mentions)
Ab235094, by Abcam (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Tnf α elisa kit, by MultiSciences Biotech (1 mentions)
Human recombinant s100a9 protein, by Abcam (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
P nf κb p65, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Sc 20067, by Santa Cruz Biotechnology (1 mentions)
Il 6 sc 57315, by Santa Cruz Biotechnology (1 mentions)
10 protocols using tlr4 sc 293072
1
Protein Expression Analysis of Colon Tissues
2
Immunohistochemical Analysis of Aortic and Intestinal Tissue
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Frozen aortic root sections adjacent to the sites of the maximum lesion area were stained with primary antibodies against CD68 (marker for macrophages, rabbit polyclonal; Abcam, catalog number ab125212), and terminal ileum tissues were stained with antibodies of zonula occludens-protein 1 (ZO-1, 33-1500, Thermo Fisher Scientific), Occludin (OCLN, 339188, Thermo Fisher Scientific), toll like receptor 4 (TLR-4, sc-293072, Santa Cruz), and toll like receptor 9 (TLR9, sc-47723, Santa Cruz) respectively, followed by their corresponding secondary antibody conjugated to horseradish peroxidase (HRP, SV0002 or SV0001, BOSTER, Wuhan, China). Staining was illuminated by Diaminobenzidine (DAB), and pictures were taken by a Leica scanning electron microscope and analyzed using Image J software.
3
Quantitative Western Blotting of Key Metabolic Regulators
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Western blotting was conducted as previously described [18 (link)]. A total of 20 μg of protein was loaded. The primary antibodies used included FXR (ab235094, Abcam PLC, Cambridge, UK), TGR5 (NBP2-23669, Novus Biologicals, Littleton, CO, USA), GPR84 (bs-13507R, Bioss, Beijing, China), TLR4 (sc-293072, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-AKT/AKT (#4060/9272s, CST, Danvers, MA, USA) and p-PI3K/PI3K (310163/R22768, ZEN-BIOSCIENCE, Chengdu, China).
4
Immunohistochemical Analysis of c-MYC Lymphoma
5
Anti-inflammatory Effects of Anemoside B4 in Colitis
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TNBS (Cat #: P2297) and lipopolysaccharide (LPS) were supplied by Sigma-Aldrich, Germany (L6529-1MG). Mesalazine (89-57-6) was purchased from Ethypharm Pharmaceutical Co Ltd (Shanghai, China). Human recombinant S100A9 protein was purchased from Abcam (AB95909). Rat IL-1β, IL-6, and TNF-α ELISA kits were supplied by MultiSciences (Hangzhou, China). PCR primers for quantitative real-time PCR analysis were synthesized by GeneWiz (Suzhou, China). The primary antibodies for p38 (8960, 1:2000 for Western blotting), p-p38 (4511, 1:1000), JNK (9252, 1:1000), p-JNK (9255, 1:2000), ERK1/2 (4695, 1:2000), p-ERK1/2 (4370, 1:1000), NF-κB/p65 (8242, 1:2000), p-NF-κB/p65 (3033, 1:1000), Bcl-2 (3498, 1:1000), cleaved-caspase 3 (9664, 1:1000), and S100A9 (73,425, 1:1000) were purchased from Cell Signaling Technology (Danvers, USA), and Bax (sc-20,067, 1:2000), p53 (sc-126, 1:1000), IL-6 (sc-57,315, 1:1000), TLR4 (sc-293,072, 1:1000), NOD2 (sc-56,168, 1:1000), and GAPDH (sc-365,062, 1:2000) were purchased from Santa Cruz Biotechnology Co. Ltd (Santa Cruz, USA). Anemoside B4 (20 g, purity more than 99%) was isolated from the roots of P. chinensis and its structure was determined as previous described [11 (link)].
6
Protein Expression Analysis in Rat Striatum
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Proteins were extracted from the rat striatum as described previously (Wang et al., 2017 (link)). The proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE). After electrophoresis, the proteins were transferred to polyvinylidine difluoride membranes. The membranes were then blocked with 5% milk-Tris-buffered solution-Tween solution for 2 h and subsequently incubated overnight at 4°C followed by appropriate secondary antibodies for 2 h at room temperature. Bands were visualized using the ECL system (BIO-RAD Laboratories, Inc., California, USA). Primary antibodies against toll-like receptor 4 (TLR4, sc-293072, Santa Cruz Biotechnology), myeloid differentiation factor 88 (MyD88, sc-74532, Santa Cruz), tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6, sc-8409, Santa Cruz), nuclear factor (NF) κB (sc-8008, Santa Cruz), interleukin (IL)-1β (sc-12742, Santa Cruz), IL-6 (sc-57315, Santa Cruz), TNF-α (sc-12744, Santa Cruz), caspase 3 (9665, Cell Signaling Technology), poly(ADP-ribose) polymerase-1 (PARP-1, sc-8007, Santa Cruz), and GAPDH (sc-32233, Santa Cruz) were used.
Densitometric analysis was conducted using Tanon Gel Image System (version 4.2). Data of relative integrated optical density values of bands are presented as bar charts.
Densitometric analysis was conducted using Tanon Gel Image System (version 4.2). Data of relative integrated optical density values of bands are presented as bar charts.
7
Western Blot Analysis of Protein Markers
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Cells were lysed via RIPA lysis buffer (Beyotime, China), and total protein concentration was measured via Pierce BCA protein analysis kit (Pierce, US). After elution in sample loading buffer (Beyotime) and separation via SDS-PAGE, the samples were transferred to PVDF films at a constant current of 200 mA. The films blocked with 5% skim milk in Tris-buffered saline Tween (TBST) at room temperature, and the primary antibodies GAPDH (2118, Cell Signaling Technology, 1:1000), p-NF-κB (3033, 1: 1000), Cell Signaling Technology), NF-κB (8242, 1:1000, Cell Signaling Technology), TLR4 (sc-293,072, 1:1000, Santa Cruz Biotechnology), Nrf2 (ab62352, 1:1000, Abcam), HO-1 (Ab13248, 1:1000, Abcam), α-SMA (A2547, 1:1000, MilliporeSigma), collagen I (ab34710, 1:1000, Abcam) were incubated. After being washed in triplicate with TBST, they were cultured with secondary antibody (goat anti-rabbit IgG, ab6721, 1:5000) in an incubator for 1.5 h at room temperature. Later being washed again with TBST 3 times, the films were treated with ECL Plus substrate bought from Life Technologies Corporation (Gaithersburg, US) to detect protein signals. The image acquisition and analysis system of Lab Works version 4.5 software (SIL Technologies, Inc., IL, US) were applied to detect and quantify the immunoreactive signal of protein bands.
8
Cardiac Protein Expression Analysis
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Cardiac tissues were homogenized, sonicated, and centrifuged at 13,000 rpm for 15 min at 4°C to remove cellular debris. Proteins (30 μg) were subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis under reduced conditions and were electroblotted onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA, USA). After blocking with 5% nonfat dry milk, the membranes were incubated with TLR4 (SC-293072) and β-MHC (MYH7, SC-53090) antibodies (1:1,000; Santa Cruz Biotechnology, Dallas, TX, USA) or anti-β-actin (SC-47778), and they were subsequently incubated with horseradish peroxidase-conjugated antimouse IgG antibody (Pierce Biotechnology, Rockford, IL, USA). The protein bands were detected using a SuperSignal substrate (Pierce Biotechnology. A densitometric analysis was performed to measure the intensity of TLR4 and MYH7 expression and β-actin bands by using AIDA software. Data were normalized to β-actin for each animal.
9
Immunohistochemical Analysis of Fibrosis Markers
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For immunohistochemistry staining, 2 μm paraffin-embedded sections were deparaffinized and rehydrated. The antigens were repaired with 1% (w/v) Tris-EDTA solution by high temperature and pressure for 10 min. Sections were blocked with BSA, then incubated with primary antibodies. The following antibodies were used: TGF-β1 (3711; Cell Signaling Technology), α-SMA (19,245; Cell Signaling Technology), fibronectin (NBP1-91258, Novus), and TLR4 (sc-293072, Santa Cruz). The secondary antibody was horseradish-peroxidase (HRP) goat anti-rabbit IgG (J31126; Transgen Biotech).
10
Proximity Ligation Assay for TLR4 and Phosphotyrosine
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The PLA was performed as described previously [16 (link)]. In short, the cells were re-hydrated, permeabilized with 0.1% Triton X (Carl Roth, Karlsruhe, Germany) and treated with 1% SDS (Carl Roth, Karlsruhe, Germany). After blocking of unspecific binding sides with Duolink Block (Sigma-Aldrich), primary antibodies were applied. For the cell culture, primary antibodies were diluted 1:200 TLR4 (sc-293072, Santa Cruz Biotechnology, Dallas, TX, USA) and 1:100 pan phospho-tyrosine antibody (p-Tyr-1000, #8954, Cell Signaling Technology, Denver, MA, USA) in antibody diluent (Sigma-Aldrich) and incubated at 4 °C over night. For the PBMCs, the antibodies were diluted 1:50 TLR4 and 1:50 p-Tyr-1000 in antibody diluent and incubated at 4 °C over night. After washing of the unbound primary antibody, the slides were incubated with proximity probes Duolink Mouse PLUS and Duolink Rabbit Minus (Sigma-Aldrich) according to the manufacturer’s instructions. After ligation and amplification using a compaction oligonucleotide described before [16 (link)], the slides were imaged in a widefield microscope.
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