Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 1 The FBG domains of tenascin-C, -R, and -W can induce NF-kB activation and cytokine synthesis, and bind to TLR4. a Tenascin-C, -R, -W, and -X each contain an assembly domain, a variable number of epidermal growth factor (EGF)-like repeats, a variable number of fibronectin type III-like repeats (these can be constitutively expressed (white rectangles) or alternatively spliced (gray rectangles) and a C-terminal fibrinogen-like globe (FBG) domain. The FBG domains exhibit a similar molecular weight, comprising between 229 and 240 amino acids each (FBG-C: 26.1 kDa, amino acids 1974–2201, FBG-R: 27.0 kDa, amino acids 1128–1359, FBG-W: 27.5 kDa, amino acids 1060–1300, FBG-X: 26.1 kDa, amino acids 4013–4243); protein accession numbers: tenascin-C (P24821), tenascin-R (Q92752), tenascin-W (Q9UQP3), tenascin-X (P22105). b THP1 NF-kB cells were stimulated with different concentrations of FBG-C, -R, -W, and -X, or were left unstimulated (−) for 24 h and NF-kB activation measured using QUANTI-Blue. Data are shown as mean ± SEM from three independent experiments. One-way ANOVA vs. non-stimulated, **p < 0.01, ***p < 0.001. c–e Primary human macrophages were stimulated with different concentrations of FBG-C,-R, -W, and -X, or were left unstimulated (−) for 24 h, and TNF (c), IL-6 (d), and IL-8 (e) levels measured by ELISA. Data are shown as mean ± SEM from three independent donors. One-way ANOVA vs. non-stimulated, *p < 0.05, **p < 0.01, ***p < 0.001. f 96-well plates were coated with 1 µg ml−1 of FBG-C, -R, -W, or -X, or PBS, and incubated with increasing doses of TLR4. Curves were fitted in GraphPad Prism using one-binding site hyperbola equation. Data in the graph are shown as mean ± SEM from four independent experiments

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques: Activation Assay, Molecular Weight, Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 2 Peptide mapping reveals specific regions in FBG-C involved in TLR4 activation and binding. a Nine peptides of ~30 amino acids long from FBG-C were synthesized; overlapping amino acid sequences are shown in bold. b THP1 NF-kB cells were stimulated with LPS (0.5 ng ml−1), FBG-C (0.5 µM), or 20, 50, or 100 µM of peptides 1–9 for 24 h and NF-kB activation was measured using QUANTI-Blue™. Data shown as mean ± SEM, n = 4 independent experiments. One-way ANOVA vs. unstimulated cells. **p < 0.01, ***p < 0.001. c Increasing doses of TLR4 were pre-incubated with 200 µM of peptides before adding them to 96-well plates coated with 1 µg ml−1 of FBG-C. Curves were fitted in GraphPad Prism using one-binding site hyperbola equation. Data are shown as mean ± SEM, n = 3. d THP1 NF-kB cells were left unstimulated (−) or pre-incubated with 100 µM peptides prior to stimulation with 0.5 µM of FBG-C for 24 h. NF-kB activation was measured using QUANTI-Blue™. Data shown as mean ± SEM, n = 3 independent experiments. Paired t-test vs. FBG- C only, *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques: Activation Assay, Binding Assay, Synthesized, Incubation

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 4 Pinpointing amino acids in loops 5, 7, and 10 of FBG-C that mediate TLR4 binding and activation. Upper panel: Sequences of wild-type FBG-C and mutants 1–7, highlighting wild-type amino acids in blue and mutations in red (loop 5 variants are shown in a, loop 10 in b, and loop 7 in c). Second panel: THP1 NF-kB cells were left unstimulated (−) or stimulated for 24 h with LPS (1 ng ml−1), increasing doses (μM) of FBG-C or FBG-C mutants 1–7. NF-kB activation was measured using QUANTI-Blue™. Data shown as mean ± SEM. n = 4 independent experiments. Paired t-test vs. FBG-C, *p < 0.05, **p < 0.01, ***p < 0.001. Third panel: Primary human macrophages were left unstimulated (−) or stimulated for 24 h with LPS (1 ng ml−1), increasing doses (μM) of FBG-C or FBG-C mutants 1–7. Cytokines synthesis was measured by ELISA. Data shown as mean ± SEM. n = 4 independent donors. Paired t-test vs. FBG-C, *p < 0.05, **p < 0.01, ***p < 0.001. Bottom panel: 96-well plates were coated with 1 µg ml−1 of FBG-C or FBG-C mutants 1–7, and TLR4 was added in a dose-dependent manner. Curves were fitted in GraphPad Prism using one-binding site hyperbola equation. Data shown as mean ± SEM; n = 4

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques: Binding Assay, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 5 Mutations in FBG-X confer TLR4-activating ability. a FBG-X chimeric proteins were designed to introduce the amino acids found in FBG-C to activate and bind to TLR4 (red) into the FBG-X sequence (blue). b ThP1 NF-kB cells were left unstimulated (−) or stimulated for 24 h with 0.5 ng ml−1 of LPS or increasing doses (μM) of FBG-C, FBG-X, FBG-X mutant 1, 2, 3, and 4. NF-kB activation was measured using QUANTI-Blue™. Data shown as mean ± SEM. n = 3 independent experiments. One-way ANOVA vs. FBG-C. c Primary human macrophages were left unstimulated (−) or stimulated for 24 h with 1 ng ml−1 of LPS or increasing doses (μM) of FBG-C, FBG-X, FBG-X mutant 1, 2, 3, and 4. Cytokine synthesis was measured by ELISA. Data shown as mean + SEM. n = 3 independent donors. One-way ANOVA vs. FBG-C. d 96-well plates were coated with 1 µg ml−1 of FBG-C, FBG-X, FBG-X mutant 1, 2, 3, and 4, and TLR4 was added in a dose-dependent manner. Data shown as mean ± SEM. n = 4 independent experiments

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques: Introduce, Sequencing, Mutagenesis, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 6 A conserved cationic ridge in fibrinogen-related proteins (FRePs). a Simplified domain organization of human FRePs: each protein contains distinct N-terminal sequences but all possess a C-terminal FBG domain, including the four tenascin family members (shown in Fig. 1a), α, β, and γ chains of fibrinogen, the three angiopoietins, seven of the angiopoietin-like proteins (Angio-LPs), the three ficolins, fibroleukin, FIBCD-1, FGL1, and MFAP4. b The cationic loop 5 ridge present in FBG-C, -R, and -W, but absent in FBG-X, is conserved in a subset of FRePs, which possess a comparable structural epitope made up of residues from loops 5, 6, and 7. Homology models of the FBG domains of the three FRePs selected for further analysis are shown together with that of tenascin-C (FBG-C); these include two predicted TLR4 agonists; the fibrinogen γ chain (FIB-G) and ficolin-1 (FIC-1), and one FBG domain predicted to be incapable of activating TLR4; angiopoietin-like protein 4 (ALP-4). The region created by residues from loops 5, 6, and 7 on the surface of each FBG domain is shown in pale orange, within which positively charged residues are colored red

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques:

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 7 The FBG domains of FIB-G and FIC-1 exhibit pro-inflammatory effects in vitro and in vivo. a–c Primary human macrophages were stimulated with different concentrations of FBG-C, FIB-G, FIC-1, and ALP-4, or were left unstimulated (−) for 24 h. Cytokine levels were measured by ELISA. Data shown as mean ± SEM from at least three independent donors. One-way ANOVA vs. non-stimulated, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. d Primary human macrophages were pre-incubated for 6 h with 3 µM TAK 242 prior to stimulation with FBG-C, FIB-G, FIC-1, and ALP-4 (1 µM), or no stimulation (−) for 24 h. Cytokine synthesis was measured by ELISA. Data shown as mean ± SEM from at least three independent donors. Paired t-test vs. non-treated, *p < 0.05, **p < 0.01, ***p < 0.001. e 96-well plates were coated with 1 µg ml−1 of FBG-C, FBG-X, FIB-G, FIC-1, and ALP-4, or PBS, and incubated with increasing doses of TLR4. Curves were fitted in GraphPad Prism using one-binding site hyperbola equation. Data are shown as mean ± SEM from three independent experiments. f, g Synovial inflammation was assessed 3 days post injection of each protein (1 µg) or PBS alone into the knees of DBA-1 mice. The histological score was calculated as the mean of seven sections from each knee joint per mouse. n = 5 mice per group except for FIC-1 (n = 4) (f). Mann–Whitney non-parametric test vs. PBS, *p < 0.05, **p < 0.01. Images show representative sections stained by haematoxylin and eosin (left panels) or safranin-O (right panels) (g). Mice injected with FBG-C, FIB-G, and FIC-1 exhibit cell infiltration into a thickened synovial lining layer, cellular invasion into the subchondral bone (arrows indicate bone erosion) and loss of articular cartilage proteoglycan (cp), pathological features not observed in mice injected with FBG-C mut or ALP-4.Scale bar left panels: 100 μM, right panels: 50 μM

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques: In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay, Injection, MANN-WHITNEY, Staining

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 8 A common danger domain revealed. Three distinct sites within the FBG domain of tenascin-C contribute to TLR4 activation (center panel); a cationic ridge made up of residues from loops 5–7 (pale orange with positive residues highlighted red), underneath which sits a triad of hydrophobic/polar residues from loop 7 (green, purple, and blue), plus a C-terminal cationic tail in loop 10 (positive residues highlighted red). The cationic ridge is the dominant inflammatory epitope; its deletion renders inflammatory stimuli inert and its ectopic expression can convert immunologically inactive proteins into TLR4 agonists. In addition to tenascin-C, in other proteins that contain FBG domains, possession of this inflammatory epitope also confers TLR4-activating capabilities, irrespective of protein family (*denotes validated domains). Together, these data reveal a common mechanism by which distinct inflammatory triggers, spanning a wide range of tissue locations, induced in response to a spectrum of different threats, can activate TLR4 to raise an immune response

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques: Activation Assay, Expressing

Journal: Molecular Medicine

Article Title: Deficiency of mindin reduces renal injury after ischemia reperfusion

doi: 10.1186/s10020-022-00578-2

Figure Lengend Snippet: Mindin deficiency inhibited TLR4/JNK/NF-κB signaling activation in renal IR injury A TLR4, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα measured by western blot analysis in kidneys of WT and mindin KO mice induced by IR or not. B – E Quantitative analysis of TLR4, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα protein expression. *P < 0.05 versus WT Sham or KO Sham. # P < 0.05 versus WT IR

Article Snippet: Mindin protein (HY-P70142, MedChemExpress) and TLR4 protein (HY-P73586, MedChemExpress) were used to study the protein binding in CM5 electronic chips by biomolecular interaction instrument (Biacore T2000).

Techniques: Activation Assay, Western Blot, Expressing

Journal: Molecular Medicine

Article Title: Deficiency of mindin reduces renal injury after ischemia reperfusion

doi: 10.1186/s10020-022-00578-2

Figure Lengend Snippet: Mindin overexpression promoted production of inflammatory mediators and activation of TLR4/JNK/ NF-κB signaling after HR in vitro. A Representative photomicrographs showing the immunofluorescence of GFP in adGFP and adMindin cells. B Mindin protein expression in adGFP and adMindin cells. C – E Protein levels of TNF-α and MCP-1 in the media of cultured TECs by Western blot. *P < 0.05 versus adGFP Con or adMindn Con. # P < 0.05 versus adGFP HR. F TLR4, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα measured by western blot analysis in adGFP and adMindin cells induced by HR or not. G – J Quantitative analysis of TLR4, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα protein expression. *P < 0.05 versus adGFP Con or adMindn Con. # P < 0.05 versus adGFP HR. K Quantitative analysis of TLR4, JNK, P65, TNF-α and MCP-1 mRNA expression in mindin-overexpressed HK-2 cells. ***P < 0.001 versus adGFP HR group

Article Snippet: Mindin protein (HY-P70142, MedChemExpress) and TLR4 protein (HY-P73586, MedChemExpress) were used to study the protein binding in CM5 electronic chips by biomolecular interaction instrument (Biacore T2000).

Techniques: Over Expression, Activation Assay, In Vitro, Immunofluorescence, Expressing, Cell Culture, Western Blot

Journal: Molecular Medicine

Article Title: Deficiency of mindin reduces renal injury after ischemia reperfusion

doi: 10.1186/s10020-022-00578-2

Figure Lengend Snippet: Mindin knockdown inhibited the activation of TLR4/JNK/ NF-κB signaling after HR in vitro. A TLR4, Mindin, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα measured by western blot analysis in si-Mindin, si-TLR4 and si-JNK cells induced by HR respectively. B – F Quantitative analysis of TLR4, Mindin, phosphorylated JNK, phosphorylated P65, and phosphorylated IκBα protein expression. * P < 0.05, ** P < 0.01 versus HR. G Quantitative analysis of TLR4, JNK, P65, TNF-α and MCP-1 mRNA expression in si-Mindin, si-TLR4 and si-JNK cells induced by HR respectively. ***P < 0.001 versus control HR group

Article Snippet: Mindin protein (HY-P70142, MedChemExpress) and TLR4 protein (HY-P73586, MedChemExpress) were used to study the protein binding in CM5 electronic chips by biomolecular interaction instrument (Biacore T2000).

Techniques: Knockdown, Activation Assay, In Vitro, Western Blot, Expressing, Control

Journal: Molecular Medicine

Article Title: Deficiency of mindin reduces renal injury after ischemia reperfusion

doi: 10.1186/s10020-022-00578-2

Figure Lengend Snippet: Co-immunoprecipitation of TLR4 and mindin in HK-2 cells. Lysates of HK-2 cells were incubated with anti-TLR4 antibody and immune complexes were precipitated by protein A/G beads. The TLR4 complex was determined TLR4 and mindin proteins in the complex. The whole cell lysate was used to detect TLR4 and mindin by Western blot

Article Snippet: Mindin protein (HY-P70142, MedChemExpress) and TLR4 protein (HY-P73586, MedChemExpress) were used to study the protein binding in CM5 electronic chips by biomolecular interaction instrument (Biacore T2000).

Techniques: Immunoprecipitation, Incubation, Western Blot

Journal: Molecular Medicine

Article Title: Deficiency of mindin reduces renal injury after ischemia reperfusion

doi: 10.1186/s10020-022-00578-2

Figure Lengend Snippet: Binding assy of mindin to TLR4 proteins by SPR. Different concen-trations of TLR4 protein (3.125, 6.25, 12.5, 25.0, 50.0, 100.0 nM) were performed to analyse the binding and dissociation rate constants between mindin and TLR4 proteins. The binding constant of them was calculated to evaluate protein binding

Article Snippet: Mindin protein (HY-P70142, MedChemExpress) and TLR4 protein (HY-P73586, MedChemExpress) were used to study the protein binding in CM5 electronic chips by biomolecular interaction instrument (Biacore T2000).

Techniques: Binding Assay, Protein Binding

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Basal TLR4 expression by (A) primary adult baboon lung DCs and (B) KG-1-derived DCs under steady-state condition. Cell-surface expression of TLR4 was detected by flow cytometry after staining the cells with TLR4-specific antibody. The percent number and MFI values of cells stained with TLR4-specific antibody (TLR4) are compared with isotype control antibody-stained cells (I) in M1 region. (C): Western blot showing undetectable expression of TLR4 in 5 μg cell lysate protein of KG-1-derived DCs (KG1-DC). An equal amount of cell lysate protein of HEK293 cells stably transfected with TLR4 (HEK-TLR4) served as positive control.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Expressing, Derivative Assay, Flow Cytometry, Staining, Western Blot, Stable Transfection, Transfection, Positive Control

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Localization of exogenously-added recombinant TLR4-MD2 protein by confocal microscopy and flow-cytometry. Confocal microscopic images of KG-1-derived DCs pulsed with Alexa-fluor 594-conjugated recombinant TLR4-MD2 protein for (A) 1h and (B) 4h. Vybrant DiO (green) dye stains the cytoplasm, and Hoechst 33342 (blue) dye stains the nucleus of the cell. The images were acquired using 63 X objective. (C) Flow-cytometric charts of KG-1-derived DCs pulsed with Alexa-fluor 495-conjugated recombinant TLR4-MD2 protein after 1h (dark line) and 4h (dotted line). The histogram chart of non-pulsed cells (negative control) is shown under the black area. Cells were gated in M region. Percent number of cells (and MFI values) positive for fluorescence are shown within the chart. Results are representative of two experiments.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Recombinant, Confocal Microscopy, Flow Cytometry, Derivative Assay, Negative Control, Fluorescence

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of purified native SP-A, recombinant TLR4-MD2 protein and MD2 protein on phagocytic function of KG-1-derived DCs. (A): Confocal microscopic images of KG-1-derived DCs incubated with pHrodo-labeled E. coli bioparticles for 3h. Phagocytosed bioparticles fluoresce red. Cells without phagocytosed particles and extracellular bacteria do not fluoresce. Enlarged images of a cell (shown as circle) are also shown in the figure, at different z-stack slices. (B): The extracellular bacteria that are either settled at the bottom or lie towards the top do not emit any fluorescence. These images confirm that fluorescence is of phagocytosed bioparticles. Next, KG-1-derived DCs were incubated with (C): purified baboon lung SP-A (0.2 and 2 μM), (D): recombinant TLR4-MD2 protein (0.06–0.6 μM) and functional-grade anti-human TLR4 antibody (HTA125 clone, Imgenex, CA; control reaction), (E): recombinant MD2 protein (0.02–0.2 μM), and (F): purified baboon lung SP-A (2 μM) and TLR4-MD2 protein (0.6 μM), for an hour prior to addition of pHrodo-labeled E. coli bioparticles. The phagocytic uptake of E. coli bioparticles was measured spectrofluorometrically at 550 nm excitation and 600 nm emission wavelengths. Results are mean (SEM) of three different experiments. * p<0.05 or ns: not significant as compared to basal phagocytosis.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Purification, Recombinant, Derivative Assay, Incubation, Labeling, Bacteria, Fluorescence, Functional Assay

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of simultaneous addition of purified SP-A and recombinant TLR4-MD2 protein on phagocytic function of primary (A) adult baboon lung DCs and (B) fetal baboon lung DC-precursor cells. The DCs were incubated with respective proteins for an hour prior to addition of pHrodo-labeled E. coli bioparticles. The phagocytic uptake of E. coli bioparticles was measured spectrofluorometrically. * p<0.05, ns: not significant or otherwise indicated. Results are mean (SEM) of three different experiments performed at different times.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Purification, Recombinant, Incubation, Labeling

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of purified native SP-A and recombinant TLR4-MD2 proteins on TNF-α secretion by DCs against E. coli. (A) Primary adult baboon lung DCs or (B) fetal baboon lung DC-precursor cells were incubated with effector molecules for an hour prior to addition of pHrodo-labeled E. coli bioparticles. After 3h incubation at 37°C in 5% CO2 incubator, the cell-free supernatants were collected and subjected to ELISA for measurement of TNF-α. The results are representative of two experiments performed separately in triplicate. * p<0.05, ** p<0.001, ns: not significant.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Purification, Recombinant, Incubation, Labeling, Enzyme-linked Immunosorbent Assay

Journal: Gut and Liver

Article Title: Pyrrolidine Dithiocarbamate Inhibits Nuclear Factor κB and Toll-Like Receptor 4 Expression in Rats with Acute Necrotizing Pancreatitis

doi: 10.5009/gnl14050

Figure Lengend Snippet: Toll-like receptor 4 expression in the pancreatic vascular endothelial cells. (A) Acute necrotizing pancreatitis (ANP) group, (B) ANP with pyrrolidine dithiocarbamate pretreatment group, and (C) sham-operation control group (A and B, H&E stain, ×200; C, H&E stain, ×400).

Article Snippet: TLR4 antibody and NF-κB antibody (Boster, Wuhan, China), NF-κB p65 antibody (Ebioscience, USA), BCA protein concentration Kit (Pierce, USA), reverse transcription polymerase chain reaction (RT-PCR) Kit (Gibco, USA), TRIzol (Invitrogen, USA), PCR primer and DNA ladder (Sbsgene, China).

Techniques: Expressing, Control, Staining

Journal: Gut and Liver

Article Title: Pyrrolidine Dithiocarbamate Inhibits Nuclear Factor κB and Toll-Like Receptor 4 Expression in Rats with Acute Necrotizing Pancreatitis

doi: 10.5009/gnl14050

Figure Lengend Snippet: Western blots of Toll-like receptor 4 (TLR4) and nuclear factor κB (NF-κB). The expression of both TLR4 and NF-κB was checked at 3-, 6-, and 12-hour time points in the pancreas of rats in the acute necrotizing pancreatitis (ANP) and pyrrolidine dithiocarbamate (PDTC) pretreated ANP groups. TLR4 and NF-κB expression in the sham-operated rat pancreases is also shown as controls.

Article Snippet: TLR4 antibody and NF-κB antibody (Boster, Wuhan, China), NF-κB p65 antibody (Ebioscience, USA), BCA protein concentration Kit (Pierce, USA), reverse transcription polymerase chain reaction (RT-PCR) Kit (Gibco, USA), TRIzol (Invitrogen, USA), PCR primer and DNA ladder (Sbsgene, China).

Techniques: Western Blot, Expressing