polyclonal neutralizing antibody against tlr4  (R&D Systems)

Journal: Frontiers in Pharmacology

Article Title: Alisol B 23-Acetate Ameliorates Lipopolysaccharide-Induced Intestinal Barrier Dysfunction by Inhibiting TLR4-NOX1/ROS Signaling Pathway in Caco-2 Cells

doi: 10.3389/fphar.2022.911196

Figure Lengend Snippet: Primer sequences of NOX1, TLR4 and β-actin in this study.

Article Snippet: The PVDF membrane was blocked with 5% non-fat milk for 2 h, and underwent incubation overnight at 4°C with primary antibodies, including anti-human polyclonal antibodies that recognize TLR4 (1:1,000, Invitrogen, United States), NOX-1 (1:1,000, Proteintech Group, Wuhan, China), ZO-1 (1:500, Invitrogen, United States), occludin (1:500, Invitrogen, United States) and β-actin (1:2,000, Beyotime Biotechnology, Jiangsu, China), At room temperature, to mix the primary antibodies for 1–2 h, the secondary goat anti-rabbit horseradish peroxidase (HRP)-IgG antibody (1:2,000, Affinity, United States) was added.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Alisol B 23-Acetate Ameliorates Lipopolysaccharide-Induced Intestinal Barrier Dysfunction by Inhibiting TLR4-NOX1/ROS Signaling Pathway in Caco-2 Cells

doi: 10.3389/fphar.2022.911196

Figure Lengend Snippet: AB23A effects on the TLR4 and NOX1 proteins expression induced by LPS. (A) The TLR4 protein expression level. (B) The NOX1 protein expression level. All gel images are from the same sample. β-actin was utilized repeatedly as a control image. ** p < 0.01 vs. control; ## p < 0.01 vs. LPS (10 μg/ml).

Article Snippet: The PVDF membrane was blocked with 5% non-fat milk for 2 h, and underwent incubation overnight at 4°C with primary antibodies, including anti-human polyclonal antibodies that recognize TLR4 (1:1,000, Invitrogen, United States), NOX-1 (1:1,000, Proteintech Group, Wuhan, China), ZO-1 (1:500, Invitrogen, United States), occludin (1:500, Invitrogen, United States) and β-actin (1:2,000, Beyotime Biotechnology, Jiangsu, China), At room temperature, to mix the primary antibodies for 1–2 h, the secondary goat anti-rabbit horseradish peroxidase (HRP)-IgG antibody (1:2,000, Affinity, United States) was added.

Techniques: Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: Alisol B 23-Acetate Ameliorates Lipopolysaccharide-Induced Intestinal Barrier Dysfunction by Inhibiting TLR4-NOX1/ROS Signaling Pathway in Caco-2 Cells

doi: 10.3389/fphar.2022.911196

Figure Lengend Snippet: AB23A effects on the ROS generation induced by LPS in TLR4-shRNA transfected cells. (A) The NOX1 mRNA expression level. (B) The NOX1 protein expression level. (C) AB23A effects on the ROS generation induced by LPS in NOX1 knockdown cells. ** p < 0.01 vs. control; ## p < 0.01 vs. LPS (10 μg/ml); &# p < 0.01 vs. NOX1-shRNA; && p < 0.01 vs. NOX1-shRNA + LPS (10 μg/ml).

Article Snippet: The PVDF membrane was blocked with 5% non-fat milk for 2 h, and underwent incubation overnight at 4°C with primary antibodies, including anti-human polyclonal antibodies that recognize TLR4 (1:1,000, Invitrogen, United States), NOX-1 (1:1,000, Proteintech Group, Wuhan, China), ZO-1 (1:500, Invitrogen, United States), occludin (1:500, Invitrogen, United States) and β-actin (1:2,000, Beyotime Biotechnology, Jiangsu, China), At room temperature, to mix the primary antibodies for 1–2 h, the secondary goat anti-rabbit horseradish peroxidase (HRP)-IgG antibody (1:2,000, Affinity, United States) was added.

Techniques: shRNA, Transfection, Expressing, Knockdown, Control

Journal: Frontiers in Pharmacology

Article Title: Alisol B 23-Acetate Ameliorates Lipopolysaccharide-Induced Intestinal Barrier Dysfunction by Inhibiting TLR4-NOX1/ROS Signaling Pathway in Caco-2 Cells

doi: 10.3389/fphar.2022.911196

Figure Lengend Snippet: AB23A effects on the LPS-induced intestinal barrier permeability by inhibiting the TLR4-NOX1/ROS pathway. Following transfection with TLR4 shRNA, the cells were subsequently cultured for 48 h before being treated with LPS for 12 h. (A,B) The mRNA and protein expression level of TLR4. (C,D) The occludin and ZO-1 protein expression level. (E) The NOX1 protein expression level after transfected with TLR4-shRNA with or without LPS stimulation. (F) The ROS production following transfection with TLR4-shRNA with or without LPS stimulation. All gel images are from the same sample. β-actin was utilized repeatedly as a control image. ** p < 0.01 vs. control; # p < 0.05 vs. TLR4-shRNA + LPS (10 μg/ml); ## p < 0.01 vs. TLR4-shRNA + LPS (10 μg/ml).

Article Snippet: The PVDF membrane was blocked with 5% non-fat milk for 2 h, and underwent incubation overnight at 4°C with primary antibodies, including anti-human polyclonal antibodies that recognize TLR4 (1:1,000, Invitrogen, United States), NOX-1 (1:1,000, Proteintech Group, Wuhan, China), ZO-1 (1:500, Invitrogen, United States), occludin (1:500, Invitrogen, United States) and β-actin (1:2,000, Beyotime Biotechnology, Jiangsu, China), At room temperature, to mix the primary antibodies for 1–2 h, the secondary goat anti-rabbit horseradish peroxidase (HRP)-IgG antibody (1:2,000, Affinity, United States) was added.

Techniques: Permeability, Transfection, shRNA, Cell Culture, Expressing, Control

Journal: Frontiers in Nutrition

Article Title: Galactooligosaccharide Treatment Alleviates DSS-Induced Colonic Inflammation in Caco-2 Cell Model

doi: 10.3389/fnut.2022.862974

Figure Lengend Snippet: Inhibition of DSS induced-NF-kB signaling pathway by Bimuno GOS in Caco-2 cells. Cells were untreated, or treated with 2% DSS alone for 6 h, or treated with 2% DSS for 2 h and then with Bimuno GOS (100 or 200 μg/ml) for a further 4 h, or treated with 100 or 200 μg/ml Bimuno GOS alone for 4 h. Protein expression was analyzed by Western blot. The relative expression levels of TLR4, MyD88, Tollip, and IRAK-M were normalized to α-tubulin, whereas the phosphorylated IKK-α, IKK-β, IkB-α, and p65 were normalized to their corresponding unphosphorylated forms. (A) TLR4, MyD88, P-IKK-α, P-IKK-β, P-IkB-α, P-p65 (densitometric values). (B) Tollip and IRAK-M (densitometric values). (C) Representative Western blot of the analyzed proteins. Values represent means ± SD of three independent experiments, carried out in triplicate. Means without a common letter significantly differ, P < 0.05.

Article Snippet: Membranes were incubated with the following primary antibodies: rabbit polyclonal anti-human TLR4, MyD88, IKKα, IKKβ, phospho(P)-IKKα/β, IkBα, P-IkBα, NF-kB p65, P-p65, IRAK-M, Tollip, from Cell Signaling Technology (Danvers, MA), mouse monoclonal α-tubulin.

Techniques: Inhibition, Expressing, Western Blot

Journal: Frontiers in Nutrition

Article Title: Galactooligosaccharide Treatment Alleviates DSS-Induced Colonic Inflammation in Caco-2 Cell Model

doi: 10.3389/fnut.2022.862974

Figure Lengend Snippet: Inhibition of DSS induced-P-p65 translocation by Bimuno GOS in Caco-2 cells. Cells were untreated (C), or treated with 2% DSS for 6 h, or treated with Bimuno GOS (100 or 200 μg/ml) for 4 h, or treated with 2% DSS for 2 h and then with Bimuno GOS (100 or 200 μg/ml) for further 4 h. Cell nuclei were stained with DAPI, while P-p65 was labeled with rabbit polyclonal anti-P-p65 antibody, followed by TRITC-conjugated secondary antibody. Each figure is representative of three independent assays (63 × magnification). Bars represent 10 μm.

Article Snippet: Membranes were incubated with the following primary antibodies: rabbit polyclonal anti-human TLR4, MyD88, IKKα, IKKβ, phospho(P)-IKKα/β, IkBα, P-IkBα, NF-kB p65, P-p65, IRAK-M, Tollip, from Cell Signaling Technology (Danvers, MA), mouse monoclonal α-tubulin.

Techniques: Inhibition, Translocation Assay, Staining, Labeling

Journal: Journal of medicinal chemistry

Article Title: Systematic Development of Peptide Inhibitors Targeting the CXCL12/HMGB1 Interaction.

doi: 10.1021/acs.jmedchem.1c00852

Figure Lengend Snippet: Figure 4. HMGB1-induced release of IL-6 and TNF via TLR4 is not inhibited by HBP08. The concentration of IL-6 (A) and TNF (B) in the supernatant of monocytes treated with HMGB1 in the presence of HBP08 or a neutralizing antibody against TLR4 (αTLR4) was measured by CBA. Data are shown as the mean ± SEM of at least four independent experiments performed. ***p < 0.001; ****p < 0.0001 by one-way ANOVA, followed by Dunnett’s multicomparison test.

Article Snippet: A polyclonal neutralizing antibody against TLR4 (AF1478, R&D System) was used to block TLR4 engagement.

Techniques: Concentration Assay

Journal: Neural Regeneration Research

Article Title: High mobility group box 1 mediates inflammatory response of astrocytes via cyclooxygenase 2/prostaglandin E2 signaling following spinal cord injury

doi: 10.4103/1673-5374.303039

Figure Lengend Snippet: A reconstructed gene network was created using the Ingenuity Pathway Analysis software on the basis of integrated differentially expressed genes involved in inflammatory responses. The different circles represent gene weights, while the arrows indicate interaction relationships. HMGB1: High mobility group box 1; MYD88: myeloid differentiation factor 88; Ptges: prostaglandin E synthase; TLR4: Toll-like receptor 4.

Article Snippet: Sections were co-incubated with mouse anti-glial fibrillary acidic protein (GFAP) polyclonal antibody (1:500; Cat# G3893; Sigma, St. Louis, MO, USA) and human anti-TLR4 polyclonal antibody (1:40; Cat# AF1478; R&D) or rabbit anti-COX2 polyclonal antibody (1:200; Cat# 160126; Cayman) at 4°C for 24 hours.

Techniques: Software

Journal: Neural Regeneration Research

Article Title: High mobility group box 1 mediates inflammatory response of astrocytes via cyclooxygenase 2/prostaglandin E2 signaling following spinal cord injury

doi: 10.4103/1673-5374.303039

Figure Lengend Snippet: Protein expression changes in HMGB1, TLR4, COXs, and PGESs in the injured spinal cord of rats following spinal cord injury. (A) Western blot analysis of HMGB1, TLR4, COX1, COX2, mPGES-1, mPGES-2, and cPGES following spinal cord contusion at 0, 1, 4, and 7 days. β-Actin was used as an internal control. (B–H) Quantification data of relative expression of HMGB1 (B), TLR4 (C), COX1 (D), COX2 (E), mPGES-1 (F), mPGES-2 (G), and cPGES (H). Relative expression is expressed as the optical density ratio to β-actin. Experiments were performed in triplicate. Data are expressed as the mean ± SEM ( n = 6). * P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc test). COX: Cyclooxygenase; cPGES: cytosolic prostaglandin E synthase; HMGB1: high mobility group box 1; mPGES: microsomal prostaglandin E synthase; TLR4: Toll-like receptor 4.

Article Snippet: Sections were co-incubated with mouse anti-glial fibrillary acidic protein (GFAP) polyclonal antibody (1:500; Cat# G3893; Sigma, St. Louis, MO, USA) and human anti-TLR4 polyclonal antibody (1:40; Cat# AF1478; R&D) or rabbit anti-COX2 polyclonal antibody (1:200; Cat# 160126; Cayman) at 4°C for 24 hours.

Techniques: Expressing, Western Blot, Control

Journal: Neural Regeneration Research

Article Title: High mobility group box 1 mediates inflammatory response of astrocytes via cyclooxygenase 2/prostaglandin E2 signaling following spinal cord injury

doi: 10.4103/1673-5374.303039

Figure Lengend Snippet: Co-localization of TLR4 and COX2 with astrocytes in the injured spinal cord of rats following SCI. (A) Illustration of section sites showing immunofluorescence. The rectangle indicates the observed field. (B) Immunofluorescence analysis of TLR4 (red, stained by Cy3) co-localization with GFAP-positive cells (green, stained by fluorescein isothiocyanate) in the injured spinal segments following SCI at 0 and 4 days. (C) Immunofluorescence analysis of COX2 (red, stained by Cy3) co-localization with GFAP-positive cells (green, stained by fluorescein isothiocyanate) in injured spinal segments following SCI at 0 and 4 days. The rectangle indicates the magnified region. Scale bars: 50 μm. COX2: Cyclooxygenase 2; GFAP: glial fibrillary acidic protein; SCI: spinal cord injury; TLR4: Toll-like receptor 4.

Article Snippet: Sections were co-incubated with mouse anti-glial fibrillary acidic protein (GFAP) polyclonal antibody (1:500; Cat# G3893; Sigma, St. Louis, MO, USA) and human anti-TLR4 polyclonal antibody (1:40; Cat# AF1478; R&D) or rabbit anti-COX2 polyclonal antibody (1:200; Cat# 160126; Cayman) at 4°C for 24 hours.

Techniques: Immunofluorescence, Staining

Journal: Neural Regeneration Research

Article Title: High mobility group box 1 mediates inflammatory response of astrocytes via cyclooxygenase 2/prostaglandin E2 signaling following spinal cord injury

doi: 10.4103/1673-5374.303039

Figure Lengend Snippet: Effects of rHMGB1 on the expression of PGE2 synthesis-related proteins in astrocytes. (A) Purified primary astrocytes stained with GFAP (green, stained by fluorescein isothiocyanate) and Hoechst 33342 (blue). (B) Western blot analysis of COX1, COX2, mPGES-1, mPGES-2, and cPGES in astrocytes following incubation with different concentrations of rHMGB1 for 24 hours. β-Actin was used as an internal control. (C–G) Quantification data of relative expression of COX1 (C), COX2 (D), mPGES-1 (E), mPGES-2 (F), and cPGES (G). The relative expression is expressed as the optical density ratio to β-actin. Experiments were performed in triplicate. Data are expressed as the mean ± SEM. * P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc test). COX: Cyclooxygenase; cPGES: cytosolic prostaglandin E synthase; GFAP: glial fibrillary acidic protein; mPGES: microsomal prostaglandin E synthase; PGE2: prostaglandin E2; rHMGB1: recombinant high mobility group box 1; TLR4: Toll-like receptor 4.

Article Snippet: Sections were co-incubated with mouse anti-glial fibrillary acidic protein (GFAP) polyclonal antibody (1:500; Cat# G3893; Sigma, St. Louis, MO, USA) and human anti-TLR4 polyclonal antibody (1:40; Cat# AF1478; R&D) or rabbit anti-COX2 polyclonal antibody (1:200; Cat# 160126; Cayman) at 4°C for 24 hours.

Techniques: Expressing, Purification, Staining, Western Blot, Incubation, Control, Recombinant

Journal: Neural Regeneration Research

Article Title: High mobility group box 1 mediates inflammatory response of astrocytes via cyclooxygenase 2/prostaglandin E2 signaling following spinal cord injury

doi: 10.4103/1673-5374.303039

Figure Lengend Snippet: Effects of TLR2/4 inhibitors on HMGB1-induced COX2 activation and PGE2 production in astrocytes. Primary cultured astrocytes were stimulated with 500 ng/mL rHMGB1 in the presence of 10 nM AO-I (TLR4 antagonist) or 10 nM C29 (TLR2 inhibitor) for 24 hours. The vehicle (0.1% dimethyl sulfoxide) was used as a control. (A) The COX2 protein level was determined by a western blot assay. (B) Quantification data of COX2 as shown in A. (C, D) PGE2 production in the lysate (C) and supernatant (D) was determined by an enzyme-linked immunosorbent assay. Experiments were performed in triplicate. Data are expressed as the mean ± SEM. * P < 0.05 (one-way analysis of variance followed by Tukey’s pos hoc test). AO-I: Atractylenolide I; COX2: cyclooxygenase 2; HMGB1: high mobility group box 1; PGE2: prostaglandin E2; rHMGB1: recombinant high mobility group box 1; TLR: Toll-like receptor.

Article Snippet: Sections were co-incubated with mouse anti-glial fibrillary acidic protein (GFAP) polyclonal antibody (1:500; Cat# G3893; Sigma, St. Louis, MO, USA) and human anti-TLR4 polyclonal antibody (1:40; Cat# AF1478; R&D) or rabbit anti-COX2 polyclonal antibody (1:200; Cat# 160126; Cayman) at 4°C for 24 hours.

Techniques: Activation Assay, Cell Culture, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant