Journal: Animals : an Open Access Journal from MDPI

Article Title: Increased Expression of Toll-Like Receptor 4 in Skin of Dogs with Discoid Lupus Erythematous (DLE)

doi: 10.3390/ani11041044

Figure Lengend Snippet: Immunohistochemical analysis for TLR4: normal epidermis with a low and diffuse intensity expression; ( A ) scale bar 75 µm; ( B ) scale bar 25 µm.

Article Snippet: Sections were pre-treated with citrate buffer for antigen-retrieval, and the labelling expression of TLR4 was evaluated using a mouse monoclonal antibody anti-TLR4, clone 76B357.1, developed against a portion of 100–200 amino acids of human TLR4 (NP_612564, Novus Biologicals, Milan, Italy) diluted 1:100.

Techniques: Immunohistochemical staining, Expressing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Increased Expression of Toll-Like Receptor 4 in Skin of Dogs with Discoid Lupus Erythematous (DLE)

doi: 10.3390/ani11041044

Figure Lengend Snippet: Immunohistochemical analysis for TLR4: four different cases of DLE with epidermis diffusely positive; ( A ) moderate intensity staining of the immunolabeled; ( B – D ) high-intensity staining of inflammatory cells. Diffuse immunolabelling of inflammatory cells at dermo-epidermal junction. ( A – D ) scale bar 50 µm.

Article Snippet: Sections were pre-treated with citrate buffer for antigen-retrieval, and the labelling expression of TLR4 was evaluated using a mouse monoclonal antibody anti-TLR4, clone 76B357.1, developed against a portion of 100–200 amino acids of human TLR4 (NP_612564, Novus Biologicals, Milan, Italy) diluted 1:100.

Techniques: Immunohistochemical staining, Staining, Immunolabeling

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: Journal of Virology

Article Title: Adenovirus Modulates Toll-Like Receptor 4 Signaling by Reprogramming ORP1L-VAP Protein Contacts for Cholesterol Transport from Endosomes to the Endoplasmic Reticulum

doi: 10.1128/JVI.01904-16

Figure Lengend Snippet: Description of antibodies used in the study a

Article Snippet: Antibodies and reagents used in the study are described in and , respectively. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Target Source Vendor Catalog no. Actin Rabbit Sigma (St. Louis, MO) A2066 EGFR Rabbit Fitzgerald (Concord, MA) 20R-ER004 FLAG Mouse Sigma F3165 GFP Mouse Stressgen (San Diego, CA) SAB-500 HRS Rabbit Bethyl (Montgomery, TX) A300-989A Lamin B1 Rabbit Abcam (Cambridge, MA) ab16048 LAMP1 Mouse DSHB b H4A3 LAMP2 Mouse DSHB H4B NF-κB (p65) Rabbit Cell Signaling (Beverly, MA) 8242 NF-κB (pSer536) Rabbit Cell Signaling 3033 ORP1L Mouse Santa Cruz (San Diego, CA) sc-376602 ORP1L Rabbit Abcam ab203352 ORP5 Rabbit Novus (Littleton, CO) NBP2-19683 Rab5 Mouse BD Biosciences (San Jose, CA) 610281 Rab7 Rabbit Santa Cruz sc6563 SREBP1 Mouse Novus NB600-582 TLR4 Mouse R&D Systems (Minneapolis, MN) MAB14782 TfR Mouse Zymed (South San Francisco, CA) 13-6800 Tubulin Rabbit Cell Signaling 2125 VAP-A Rabbit Sigma HPA009174 VAP-B Mouse Proteintech (Rosemont, IL) 14477-1-AP Open in a separate window a Antibodies were routinely used at a dilution of 1:1,000 to 1:2,000 for immunoblotting and at a dilution of 1:100 to 1:500 for immunostaining. b DSHB, Developmental Studies Hybridoma Bank (developed under the auspices of the Eunice Kennedy Shriver National Institute of Child Health and Human Development [NICHD] and maintained by The University of Iowa Department of Biological Sciences [Iowa City, IA]).

Techniques: