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mouse anti human tlr4 monoclonal antibody (Santa Cruz Biotechnology)

Name: mouse anti human tlr4 monoclonal antibody
Brand: Santa Cruz Biotechnology
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Santa Cruz Biotechnology mouse anti human tlr4 monoclonal antibody

The proinflammatory activity of IFI16 lies within its N-terminal region (A) qRT-PCR analysis for TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h with IFI16 alone (10 μg/mL) or pre-incubated for 1 h with the indicated amounts of anti-IFI16 polyclonal antibodies directed against either the N- or C-terminal region of the protein. Values are normalized to GAPDH mRNA and plotted as fold of induction over untreated cells. qRT-PCR data are presented as mean values of biological triplicates. Error bars indicate SEM (∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test). (B) Protein concentration of TNF-α and IL-8 determined by ELISA in the culture supernatants harvested from human macrophages stimulated for 24 h as described in (A). Data are expressed as mean values ±SEM of three independent experiments (∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test). (C) Surface plasmon resonance (SPR) analysis of IFI16 binding to immobilized <t>TLR4.</t> 500 nM of IFI16 diluted in running buffer, alone or pre-incubated for 1 h at RT with increasing concentrations (62.5–500 nM) of the anti-N-term-IFI16 antibody (red bars) or with 500 nM of anti-C-term antibody (green bar), were flowed over a TLR4/MD2-coated chip. Data are representative of two independent experiments, shown as the mean ± SEM (∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test).

Mouse Anti Human Tlr4 Monoclonal Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members"

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

Journal: iScience

doi: 10.1016/j.isci.2025.112413

Figure Legend Snippet: The proinflammatory activity of IFI16 lies within its N-terminal region (A) qRT-PCR analysis for TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h with IFI16 alone (10 μg/mL) or pre-incubated for 1 h with the indicated amounts of anti-IFI16 polyclonal antibodies directed against either the N- or C-terminal region of the protein. Values are normalized to GAPDH mRNA and plotted as fold of induction over untreated cells. qRT-PCR data are presented as mean values of biological triplicates. Error bars indicate SEM (∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test). (B) Protein concentration of TNF-α and IL-8 determined by ELISA in the culture supernatants harvested from human macrophages stimulated for 24 h as described in (A). Data are expressed as mean values ±SEM of three independent experiments (∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test). (C) Surface plasmon resonance (SPR) analysis of IFI16 binding to immobilized TLR4. 500 nM of IFI16 diluted in running buffer, alone or pre-incubated for 1 h at RT with increasing concentrations (62.5–500 nM) of the anti-N-term-IFI16 antibody (red bars) or with 500 nM of anti-C-term antibody (green bar), were flowed over a TLR4/MD2-coated chip. Data are representative of two independent experiments, shown as the mean ± SEM (∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test).

Techniques Used: Activity Assay, Quantitative RT-PCR, Expressing, Incubation, Protein Concentration, Enzyme-linked Immunosorbent Assay, SPR Assay, Binding Assay

The PYRIN domain of IFI16 is involved in TLR4/MD2 binding and activation (A) Schematic representation of the full-length IFI16 (IFI16 FL ), the truncated forms IFI16ΔHINB and IFI16ΔPYD, and the single domains used in this study. Numbers represent the amino acid positions based on the NCBI Reference Sequence GenBank: NP_005522 . (B) qRT-PCR analysis of TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h with or without the recombinant proteins described in A (111 nM). Values are normalized to GAPDH mRNA and plotted as fold induction over mock-treated cells. qRT-PCR data are presented as mean values ±SEM of biological triplicates (∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (C) Protein concentration of TNF-α and IL-8 evaluated by ELISA in supernatants derived from human macrophages stimulated for 24 h as described in the legend (B), with or without CLI-095 (TLR4 inhibitor, 5μM). Data are expressed as mean values ±SEM of three independent experiments (∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (D) SPR analysis of IFI16 mutants or domains binding to immobilized TLR4/MD2. After immobilization of TLR4/MD2 on the CM5 sensor chip surface, increasing concentration of IFI16 FL , IFI16ΔHINB, IFI16ΔPYD, IFI16 PYD , HINA, or HINB domains (20–800 nM), diluted in running buffer, were injected over the immobilized complex. Data are representative of three different experiments, with the dissociation constant (K D ) value provided where applicable. In the sensogram for IFI16ΔPYD, the asterisk (∗) denotes the mass transport effect observed at the highest concentration (2 μM). (E) Human macrophages were stimulated for 1 h with IFI16 FL , IFI16 PYD , IFI16ΔPYD, or left untreated (25 μg/mL). Total cellular extracts were then subjected to immunoprecipitation using an anti-TLR4 monoclonal antibody. Immunoprecipitates (left, IP:TLR4) and whole-cell lysates (right, Input) were analyzed by immunoblotting using antibodies against N-term-IFI16, C-term-IFI16 or TLR4. β-actin protein expression served as a protein loading control. Data are representative of three independent experiments with similar results.

Figure Legend Snippet: The PYRIN domain of IFI16 is involved in TLR4/MD2 binding and activation (A) Schematic representation of the full-length IFI16 (IFI16 FL ), the truncated forms IFI16ΔHINB and IFI16ΔPYD, and the single domains used in this study. Numbers represent the amino acid positions based on the NCBI Reference Sequence GenBank: NP_005522 . (B) qRT-PCR analysis of TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h with or without the recombinant proteins described in A (111 nM). Values are normalized to GAPDH mRNA and plotted as fold induction over mock-treated cells. qRT-PCR data are presented as mean values ±SEM of biological triplicates (∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (C) Protein concentration of TNF-α and IL-8 evaluated by ELISA in supernatants derived from human macrophages stimulated for 24 h as described in the legend (B), with or without CLI-095 (TLR4 inhibitor, 5μM). Data are expressed as mean values ±SEM of three independent experiments (∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (D) SPR analysis of IFI16 mutants or domains binding to immobilized TLR4/MD2. After immobilization of TLR4/MD2 on the CM5 sensor chip surface, increasing concentration of IFI16 FL , IFI16ΔHINB, IFI16ΔPYD, IFI16 PYD , HINA, or HINB domains (20–800 nM), diluted in running buffer, were injected over the immobilized complex. Data are representative of three different experiments, with the dissociation constant (K D ) value provided where applicable. In the sensogram for IFI16ΔPYD, the asterisk (∗) denotes the mass transport effect observed at the highest concentration (2 μM). (E) Human macrophages were stimulated for 1 h with IFI16 FL , IFI16 PYD , IFI16ΔPYD, or left untreated (25 μg/mL). Total cellular extracts were then subjected to immunoprecipitation using an anti-TLR4 monoclonal antibody. Immunoprecipitates (left, IP:TLR4) and whole-cell lysates (right, Input) were analyzed by immunoblotting using antibodies against N-term-IFI16, C-term-IFI16 or TLR4. β-actin protein expression served as a protein loading control. Data are representative of three independent experiments with similar results.

Techniques Used: Binding Assay, Activation Assay, Sequencing, Quantitative RT-PCR, Expressing, Recombinant, Protein Concentration, Enzyme-linked Immunosorbent Assay, Derivative Assay, Concentration Assay, Injection, Immunoprecipitation, Western Blot, Control

The proinflammatory activity of the PYRIN domain is conserved among PYHIN family members and across species Equimolar concentrations (111 nM) of the PYDs from various human and mouse family members, as well as from the unrelated NLRP3 and ASC proteins, were used to stimulate human (A and B) or mouse (C) macrophages for 24 h, with or without CLI-095 (TLR4 inhibitor, 5 μM). (A) qRT-PCR analysis of TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h. Values are normalized to GAPDH mRNA and plotted as fold induction over untreated cells. qRT-PCR data are presented as mean values ±SEM of biological triplicates (∗∗∗ p < 0.001, ∗∗ p < 0.01; one-way ANOVA followed by Dunnett’s test). (B and C) Protein concentration of TNF-α and IL-8 in the supernatants were evaluated by ELISA. Data are representative of three independent experiments, shown as the mean ± SEM (∗∗∗ p < 0.001, ### p < 0.001; one-way ANOVA followed by Dunnett’s test). (D) SPR analysis showing the binding of various PYDs to TLR4/MD2. The TLR4/MD2 complex was immobilized on a CM5 sensor chip surface, followed by the injection of increasing concentrations of the indicated PYDs (20–800 nM) in running buffer. Data are representative of three different experiments, and for each analysis the dissociation constant (K D ) value is shown.

Figure Legend Snippet: The proinflammatory activity of the PYRIN domain is conserved among PYHIN family members and across species Equimolar concentrations (111 nM) of the PYDs from various human and mouse family members, as well as from the unrelated NLRP3 and ASC proteins, were used to stimulate human (A and B) or mouse (C) macrophages for 24 h, with or without CLI-095 (TLR4 inhibitor, 5 μM). (A) qRT-PCR analysis of TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h. Values are normalized to GAPDH mRNA and plotted as fold induction over untreated cells. qRT-PCR data are presented as mean values ±SEM of biological triplicates (∗∗∗ p < 0.001, ∗∗ p < 0.01; one-way ANOVA followed by Dunnett’s test). (B and C) Protein concentration of TNF-α and IL-8 in the supernatants were evaluated by ELISA. Data are representative of three independent experiments, shown as the mean ± SEM (∗∗∗ p < 0.001, ### p < 0.001; one-way ANOVA followed by Dunnett’s test). (D) SPR analysis showing the binding of various PYDs to TLR4/MD2. The TLR4/MD2 complex was immobilized on a CM5 sensor chip surface, followed by the injection of increasing concentrations of the indicated PYDs (20–800 nM) in running buffer. Data are representative of three different experiments, and for each analysis the dissociation constant (K D ) value is shown.

Techniques Used: Activity Assay, Quantitative RT-PCR, Expressing, Protein Concentration, Enzyme-linked Immunosorbent Assay, Binding Assay, Injection

Structural analysis of the interaction moiety between the IFI16 PYD and the TLR4/MD2 complex (A) Predicted 3D structure of the IFI16 PYD using the Robetta software. (B) Molecular docking analysis of the IFI16 PYD (green) and the extracellular portion of TLR4 (blue, PDB: 3FXI ) using High Ambiguity Driven protein-protein DOCKing software. (C) Alignment of the primary sequence of PYHIN PYDs along with those of NLRP3 and ASC performed using MEGAX and ClustalW algorithm to identify PYHIN-specific amino acids with similar chemical properties. Green arrows identify amino acids conserved across the different proteins, whereas light blue arrows identify amino acids that are conserved only across the PYHIN family members, which were used in mutagenesis experiments (see <xref ref-type=

Figure 5 ). The red arrow identifies an amino acid conserved in all the proteins, which was mutated and used as control. The six α helices of the PYDs with known structures are also indicated. (D) Identification of the IFI16 PYD residues (green) potentially involved in the interaction with TLR4 (Lys34, Lys64, and Lys86) obtained by integrating the alignment and molecular docking information. " title="... interaction moiety between the IFI16 PYD and the TLR4/MD2 complex (A) Predicted 3D structure of the IFI16 ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: Structural analysis of the interaction moiety between the IFI16 PYD and the TLR4/MD2 complex (A) Predicted 3D structure of the IFI16 PYD using the Robetta software. (B) Molecular docking analysis of the IFI16 PYD (green) and the extracellular portion of TLR4 (blue, PDB: 3FXI ) using High Ambiguity Driven protein-protein DOCKing software. (C) Alignment of the primary sequence of PYHIN PYDs along with those of NLRP3 and ASC performed using MEGAX and ClustalW algorithm to identify PYHIN-specific amino acids with similar chemical properties. Green arrows identify amino acids conserved across the different proteins, whereas light blue arrows identify amino acids that are conserved only across the PYHIN family members, which were used in mutagenesis experiments (see Figure 5 ). The red arrow identifies an amino acid conserved in all the proteins, which was mutated and used as control. The six α helices of the PYDs with known structures are also indicated. (D) Identification of the IFI16 PYD residues (green) potentially involved in the interaction with TLR4 (Lys34, Lys64, and Lys86) obtained by integrating the alignment and molecular docking information.

Techniques Used: Software, Sequencing, Mutagenesis, Control

Three amino acids located within the IFI16 PYD are critically involved in its TLR4-mediated proinflammatory activity (A) Schematic representation of the polar residues identified in <xref ref-type=

Figures 4 E and 4F, highlighting their positions within the IFI16 PYD and the specific amino acid substitutions used in the mutagenesis experiments. (B) Human macrophages were stimulated for 24 h with equimolar concentrations (111 nM) of the IFI16 mutants described in (A ) and the IFI16 PYD−TM , alongside the wild type IFI16 FL . The levels of TNF-α released in the culture supernatants were measured by ELISA. Data are representative of three independent experiments, shown as the mean ± SEM (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (C) SPR analysis assessing the binding of IFI16 FL and its mutants to immobilized TLR4/MD2 on a CM5 sensor chip. Increasing concentration of IFI16 FL or the mutants (20–800 nM), diluted in running buffer, were flowed over the immobilized complex. Data are representative of three different experiments, and for each analysis the dissociation constant (K D ) value is shown. " title="... the IFI16 PYD are critically involved in its TLR4-mediated proinflammatory activity (A) Schematic representation of the polar ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: Three amino acids located within the IFI16 PYD are critically involved in its TLR4-mediated proinflammatory activity (A) Schematic representation of the polar residues identified in Figures 4 E and 4F, highlighting their positions within the IFI16 PYD and the specific amino acid substitutions used in the mutagenesis experiments. (B) Human macrophages were stimulated for 24 h with equimolar concentrations (111 nM) of the IFI16 mutants described in (A ) and the IFI16 PYD−TM , alongside the wild type IFI16 FL . The levels of TNF-α released in the culture supernatants were measured by ELISA. Data are representative of three independent experiments, shown as the mean ± SEM (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (C) SPR analysis assessing the binding of IFI16 FL and its mutants to immobilized TLR4/MD2 on a CM5 sensor chip. Increasing concentration of IFI16 FL or the mutants (20–800 nM), diluted in running buffer, were flowed over the immobilized complex. Data are representative of three different experiments, and for each analysis the dissociation constant (K D ) value is shown.

Techniques Used: Activity Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay

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Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: The proinflammatory activity of IFI16 lies within its N-terminal region (A) qRT-PCR analysis for TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h with IFI16 alone (10 μg/mL) or pre-incubated for 1 h with the indicated amounts of anti-IFI16 polyclonal antibodies directed against either the N- or C-terminal region of the protein. Values are normalized to GAPDH mRNA and plotted as fold of induction over untreated cells. qRT-PCR data are presented as mean values of biological triplicates. Error bars indicate SEM (∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test). (B) Protein concentration of TNF-α and IL-8 determined by ELISA in the culture supernatants harvested from human macrophages stimulated for 24 h as described in (A). Data are expressed as mean values ±SEM of three independent experiments (∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test). (C) Surface plasmon resonance (SPR) analysis of IFI16 binding to immobilized TLR4. 500 nM of IFI16 diluted in running buffer, alone or pre-incubated for 1 h at RT with increasing concentrations (62.5–500 nM) of the anti-N-term-IFI16 antibody (red bars) or with 500 nM of anti-C-term antibody (green bar), were flowed over a TLR4/MD2-coated chip. Data are representative of two independent experiments, shown as the mean ± SEM (∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test).

Article Snippet: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-13593; RRID: AB_628366.

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Incubation, Protein Concentration, Enzyme-linked Immunosorbent Assay, SPR Assay, Binding Assay

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: The PYRIN domain of IFI16 is involved in TLR4/MD2 binding and activation (A) Schematic representation of the full-length IFI16 (IFI16 FL ), the truncated forms IFI16ΔHINB and IFI16ΔPYD, and the single domains used in this study. Numbers represent the amino acid positions based on the NCBI Reference Sequence GenBank: NP_005522 . (B) qRT-PCR analysis of TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h with or without the recombinant proteins described in A (111 nM). Values are normalized to GAPDH mRNA and plotted as fold induction over mock-treated cells. qRT-PCR data are presented as mean values ±SEM of biological triplicates (∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (C) Protein concentration of TNF-α and IL-8 evaluated by ELISA in supernatants derived from human macrophages stimulated for 24 h as described in the legend (B), with or without CLI-095 (TLR4 inhibitor, 5μM). Data are expressed as mean values ±SEM of three independent experiments (∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (D) SPR analysis of IFI16 mutants or domains binding to immobilized TLR4/MD2. After immobilization of TLR4/MD2 on the CM5 sensor chip surface, increasing concentration of IFI16 FL , IFI16ΔHINB, IFI16ΔPYD, IFI16 PYD , HINA, or HINB domains (20–800 nM), diluted in running buffer, were injected over the immobilized complex. Data are representative of three different experiments, with the dissociation constant (K D ) value provided where applicable. In the sensogram for IFI16ΔPYD, the asterisk (∗) denotes the mass transport effect observed at the highest concentration (2 μM). (E) Human macrophages were stimulated for 1 h with IFI16 FL , IFI16 PYD , IFI16ΔPYD, or left untreated (25 μg/mL). Total cellular extracts were then subjected to immunoprecipitation using an anti-TLR4 monoclonal antibody. Immunoprecipitates (left, IP:TLR4) and whole-cell lysates (right, Input) were analyzed by immunoblotting using antibodies against N-term-IFI16, C-term-IFI16 or TLR4. β-actin protein expression served as a protein loading control. Data are representative of three independent experiments with similar results.

Article Snippet: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-13593; RRID: AB_628366.

Techniques: Binding Assay, Activation Assay, Sequencing, Quantitative RT-PCR, Expressing, Recombinant, Protein Concentration, Enzyme-linked Immunosorbent Assay, Derivative Assay, Concentration Assay, Injection, Immunoprecipitation, Western Blot, Control

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: The proinflammatory activity of the PYRIN domain is conserved among PYHIN family members and across species Equimolar concentrations (111 nM) of the PYDs from various human and mouse family members, as well as from the unrelated NLRP3 and ASC proteins, were used to stimulate human (A and B) or mouse (C) macrophages for 24 h, with or without CLI-095 (TLR4 inhibitor, 5 μM). (A) qRT-PCR analysis of TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h. Values are normalized to GAPDH mRNA and plotted as fold induction over untreated cells. qRT-PCR data are presented as mean values ±SEM of biological triplicates (∗∗∗ p < 0.001, ∗∗ p < 0.01; one-way ANOVA followed by Dunnett’s test). (B and C) Protein concentration of TNF-α and IL-8 in the supernatants were evaluated by ELISA. Data are representative of three independent experiments, shown as the mean ± SEM (∗∗∗ p < 0.001, ### p < 0.001; one-way ANOVA followed by Dunnett’s test). (D) SPR analysis showing the binding of various PYDs to TLR4/MD2. The TLR4/MD2 complex was immobilized on a CM5 sensor chip surface, followed by the injection of increasing concentrations of the indicated PYDs (20–800 nM) in running buffer. Data are representative of three different experiments, and for each analysis the dissociation constant (K D ) value is shown.

Article Snippet: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-13593; RRID: AB_628366.

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Protein Concentration, Enzyme-linked Immunosorbent Assay, Binding Assay, Injection

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: Structural analysis of the interaction moiety between the IFI16 PYD and the TLR4/MD2 complex (A) Predicted 3D structure of the IFI16 PYD using the Robetta software. (B) Molecular docking analysis of the IFI16 PYD (green) and the extracellular portion of TLR4 (blue, PDB: 3FXI ) using High Ambiguity Driven protein-protein DOCKing software. (C) Alignment of the primary sequence of PYHIN PYDs along with those of NLRP3 and ASC performed using MEGAX and ClustalW algorithm to identify PYHIN-specific amino acids with similar chemical properties. Green arrows identify amino acids conserved across the different proteins, whereas light blue arrows identify amino acids that are conserved only across the PYHIN family members, which were used in mutagenesis experiments (see Figure 5 ). The red arrow identifies an amino acid conserved in all the proteins, which was mutated and used as control. The six α helices of the PYDs with known structures are also indicated. (D) Identification of the IFI16 PYD residues (green) potentially involved in the interaction with TLR4 (Lys34, Lys64, and Lys86) obtained by integrating the alignment and molecular docking information.

Article Snippet: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-13593; RRID: AB_628366.

Techniques: Software, Sequencing, Mutagenesis, Control

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: Three amino acids located within the IFI16 PYD are critically involved in its TLR4-mediated proinflammatory activity (A) Schematic representation of the polar residues identified in Figures 4 E and 4F, highlighting their positions within the IFI16 PYD and the specific amino acid substitutions used in the mutagenesis experiments. (B) Human macrophages were stimulated for 24 h with equimolar concentrations (111 nM) of the IFI16 mutants described in (A ) and the IFI16 PYD−TM , alongside the wild type IFI16 FL . The levels of TNF-α released in the culture supernatants were measured by ELISA. Data are representative of three independent experiments, shown as the mean ± SEM (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (C) SPR analysis assessing the binding of IFI16 FL and its mutants to immobilized TLR4/MD2 on a CM5 sensor chip. Increasing concentration of IFI16 FL or the mutants (20–800 nM), diluted in running buffer, were flowed over the immobilized complex. Data are representative of three different experiments, and for each analysis the dissociation constant (K D ) value is shown.

Article Snippet: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-13593; RRID: AB_628366.

Techniques: Activity Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay

The percentage of the neutrophil extracellular trap (NET) area in the SS and FS plots of flow cytometry was compared among untreated neutrophils and samples following NET induction with E. coli DH5α or PMA. (a) Expression intensities of Cit-H3, flavocytochrome b558, SYTOX™ Green, CD14, TLR2, TLR4, and LL-37 were compared between untreated neutrophil and NET areas after E. coli DH5α stimulation. (b) NET ratios were compared among untreated neutrophils and samples treated with E. coli DH5α or PMA using enzyme-linked immunosorbent assay (ELISA). Data are presented as mean ± standard deviation (SD) (n = 3). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. ** p < 0.01. (c) Western blotting was performed to examine CD14 and Cit-H3 protein levels in untreated neutrophils and those stimulated with E. coli DH5α or PMA. Band intensities were quantified using ImageJ and normalized to β-actin. Data are presented as mean ± standard deviation (SD) (n = 3). Data are presented as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey–Kramer’s HSD test. ** p < 0.01

Journal: bioRxiv

Article Title: Proteinase 3 is involved in presepsin production through neutrophil extracellular trap phagocytosis by macrophages

doi: 10.1101/2025.05.02.651854

Figure Lengend Snippet: The percentage of the neutrophil extracellular trap (NET) area in the SS and FS plots of flow cytometry was compared among untreated neutrophils and samples following NET induction with E. coli DH5α or PMA. (a) Expression intensities of Cit-H3, flavocytochrome b558, SYTOX™ Green, CD14, TLR2, TLR4, and LL-37 were compared between untreated neutrophil and NET areas after E. coli DH5α stimulation. (b) NET ratios were compared among untreated neutrophils and samples treated with E. coli DH5α or PMA using enzyme-linked immunosorbent assay (ELISA). Data are presented as mean ± standard deviation (SD) (n = 3). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. ** p < 0.01. (c) Western blotting was performed to examine CD14 and Cit-H3 protein levels in untreated neutrophils and those stimulated with E. coli DH5α or PMA. Band intensities were quantified using ImageJ and normalized to β-actin. Data are presented as mean ± standard deviation (SD) (n = 3). Data are presented as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey–Kramer’s HSD test. ** p < 0.01

Article Snippet: Details of the antibodies used in this study and their sources are as follows: FITC-conjugated anti-human PR3 mouse monoclonal antibody (Cat. No. ab65255; Abcam, Cambridge, UK), PE-conjugated anti-human cathepsin D mouse monoclonal antibody (Cat. No. sc-13148 PE; Santa Cruz Biotechnology, Dallas, TX, USA), FITC-conjugated anti-human neutrophil elastase mouse monoclonal antibody (Cat. No. sc-55549 FITC; Santa Cruz Biotechnology, Dallas, TX, USA), PE-conjugated anti-human flavocytochrome b558 mouse monoclonal antibody (Cat. No. D162-5; Medical & Biological Laboratories, Tokyo, Japan), PE/Cyanine7-conjugated anti-human CD14 mouse monoclonal antibody (Cat. No. 367111; BioLegend, San Diego, CA, USA), FITC-conjugated anti-human CD68 mouse monoclonal antibody (Cat. No. F7135; DAKO, Glostrup, Denmark), FITC-conjugated anti-human CD282 (TLR2) mouse monoclonal antibody (Cat. No. 309705; BioLegend, San Diego, CA, USA), PE-conjugated anti-human CD284 (TLR4) mouse monoclonal antibody (Cat. No. 312805; BioLegend, San Diego, CA, USA), anti-citrullinated histone H3 (Cit-H3) (citrulline R2 + R8 + R17) rabbit polyclonal antibody (Cat. No. ab5103; Abcam, Cambridge, UK), anti-human LL-37 mouse monoclonal antibody (Cat. No. sc-166770; Santa Cruz Biotechnology, Dallas, TX, USA), anti-β-actin rabbit monoclonal antibody (Cat. No. 4970; Cell Signaling Technology, Danvers, MA, USA), anti-human CD14 mouse monoclonal antibody (Cat. No. 14-0149-82; Invitrogen, Waltham, MA, USA), anti-PR3 mouse monoclonal antibody (Cat. No. sc-74534; Santa Cruz Biotechnology, Dallas, TX, USA), anti-human presepsin mouse monoclonal antibody (F1106-13–3; Mochida Pharmaceutical, Tokyo, Japan), anti-human presepsin rabbit monoclonal antibody (S68; Mochida Pharmaceutical, Tokyo, Japan), tetramethylrhodamine (TRITC)-conjugated goat anti-mouse IgG (H+L) secondary antibody (Cat. No. SA00007-1; Cosmo Bio, Tokyo, Japan), TRITC-conjugated goat anti-rabbit IgG (H+L) secondary antibody (Cat. No. SA00007-2, Cosmo Bio, Tokyo, Japan), Alexa Flour 405-conjugated goat anti-rabbit IgG (H+L) secondary antibody (Cat. No. ab175652; Invitrogen, Waltham, MA, USA), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) secondary antibody (Cat. No. 7076; Cell Signaling Technology, Danvers, MA, USA), HRP-conjugated gort anti-rabbit IgG (H+L) secondary antibody (Cat. No. 7074; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation, Western Blot

(A) Volcano plot displaying the proteins detected in the protein corona of PAMAM5000 and PAMAM350, here denoted OA5000 and OA350 using proteomics analysis. Siglec-5 is indicated with a circle. (B) Protein-protein interaction network visualized from the IntAct database. Strong evidence of interaction appears to TLR4, PHOP2, and KLHL8, as evidenced by high confidence score. The illustration was adopted from the website and improved using R. (C) Percentage intermediate monocytes (CD14 + CD16 + ) after 24hrs in media control and SF1-7 ( n =3 from three biological replicates). (D-F) Median fluorescence intensity (MFI) of TLR4, Siglec-5, and Siglec-9 on the cell membrane ( n =3 from three biological replicates). (G-J) Correlation between Siglec-5 to TLR4 and intermediate monocytes, and between Siglec-9 to TLR4 and intermediate monocytes. (K) Representative flow cytometric dot plots displaying the ratio between classical and intermediate monocytes, where blue represents both subsets, purple represents Siglec-5 + cells, and green represents TLR4 + cells. (L) Baseline levels (0hrs) of classical, intermediate, and non-classical subsets, where blue color represents all monocytes, purple color represents Siglec-5 + , and green represents TLR4 + cells. (M-O)MFI of TLR4, Siglec-5, and Siglec-9 in monocyte subsets. Data is presented as mean percentage or MFI ( n =2-3) from three biological replicates. Data is represented as mean±SD, statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons or Dunnett’s multiple comparisons test. Correlation analysis was performed using Spearman’s correlation test (* p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001).

Journal: bioRxiv

Article Title: Sialoglycans modulate Siglec-5 – TLR4 Interactions in Osteoarthritis

doi: 10.1101/2025.03.18.643878

Figure Lengend Snippet: (A) Volcano plot displaying the proteins detected in the protein corona of PAMAM5000 and PAMAM350, here denoted OA5000 and OA350 using proteomics analysis. Siglec-5 is indicated with a circle. (B) Protein-protein interaction network visualized from the IntAct database. Strong evidence of interaction appears to TLR4, PHOP2, and KLHL8, as evidenced by high confidence score. The illustration was adopted from the website and improved using R. (C) Percentage intermediate monocytes (CD14 + CD16 + ) after 24hrs in media control and SF1-7 ( n =3 from three biological replicates). (D-F) Median fluorescence intensity (MFI) of TLR4, Siglec-5, and Siglec-9 on the cell membrane ( n =3 from three biological replicates). (G-J) Correlation between Siglec-5 to TLR4 and intermediate monocytes, and between Siglec-9 to TLR4 and intermediate monocytes. (K) Representative flow cytometric dot plots displaying the ratio between classical and intermediate monocytes, where blue represents both subsets, purple represents Siglec-5 + cells, and green represents TLR4 + cells. (L) Baseline levels (0hrs) of classical, intermediate, and non-classical subsets, where blue color represents all monocytes, purple color represents Siglec-5 + , and green represents TLR4 + cells. (M-O)MFI of TLR4, Siglec-5, and Siglec-9 in monocyte subsets. Data is presented as mean percentage or MFI ( n =2-3) from three biological replicates. Data is represented as mean±SD, statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons or Dunnett’s multiple comparisons test. Correlation analysis was performed using Spearman’s correlation test (* p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001).

Article Snippet: Detection was performed using anti-human TLR4 mouse monoclonal antibody (HTA-125, Invitrogen) and anti-human Siglec-5 rabbit polyclonal antibody (13230-1-AP, Proteintech), both diluted 1:100 and stained for 1h in room temperature.

Techniques: Control, Fluorescence, Membrane

(A-C) MFI of TLR4, Siglec-5, and Siglec-9 at 0, 4, and 24hrs of stimulation with M-CSF, IL1β/TNFɑ, LPS, and sialidase ( n = 2-3 from one biological replicate for TLR4, and three biological replicates for Siglec-5 and Siglec-9). (D) Percentage intermediate monocytes after 24hrs ( n = 2-3 from three biological replicates). (E-G) MFI of TLR4, Siglec-5, and Siglec-9 after stimulation for 24hrs ( n = 2-3 from one biological replicate for TLR4, and three biological replicates for Siglec-5 and Siglec-9). (H) Representative flow cytometric dot plots displaying the percentage classical and intermediate monocytes where blue represents all cells, purple represents Siglec-5 + cells, and green represents TLR4 + cells, which can also be displayed in the dot plot below. Data is represented as mean±SD, statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test (* p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001).

Journal: bioRxiv

Article Title: Sialoglycans modulate Siglec-5 – TLR4 Interactions in Osteoarthritis

doi: 10.1101/2025.03.18.643878

Figure Lengend Snippet: (A-C) MFI of TLR4, Siglec-5, and Siglec-9 at 0, 4, and 24hrs of stimulation with M-CSF, IL1β/TNFɑ, LPS, and sialidase ( n = 2-3 from one biological replicate for TLR4, and three biological replicates for Siglec-5 and Siglec-9). (D) Percentage intermediate monocytes after 24hrs ( n = 2-3 from three biological replicates). (E-G) MFI of TLR4, Siglec-5, and Siglec-9 after stimulation for 24hrs ( n = 2-3 from one biological replicate for TLR4, and three biological replicates for Siglec-5 and Siglec-9). (H) Representative flow cytometric dot plots displaying the percentage classical and intermediate monocytes where blue represents all cells, purple represents Siglec-5 + cells, and green represents TLR4 + cells, which can also be displayed in the dot plot below. Data is represented as mean±SD, statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test (* p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001).

Techniques:

(A) Clustering analysis was performed on concatenated monocytes stimulated with M-CSF, IL1β/TNFɑ, LPS, sialidase, and SF1-7. Subsequently, monocytes were downsampled to 100,000 cells before performing clustering. This generated six monocyte subpopulations, the table displays each population and its count, respectively. (B) Heatmap analysis demonstrate the six populations and their relative expression of TLR4, CD16, Siglec-9, Siglec-5, and CD14. (C) Each UMAP contour density plot represents one condition, i.e. M-CSF, IL1β/TNFɑ, LPS, sialidase, and SF1-7. (D) The concentration of IL-6 (pg/mL) produced by the stimulated monocytes were quantified, grey bars represent SFs and black bars represent the pathway-specific stimulations. The mean concentration is observed in each bar (D). Data is represented as mean±SD. For the monocytes stimulated with SF1-5, three technical replicates were used from three biological replicates. For monocytes stimulated with M-CSF, IL1β/TNFɑ, LPS, sialidase, three technical replicates were used from one biological replicate.

Journal: bioRxiv

Article Title: Sialoglycans modulate Siglec-5 – TLR4 Interactions in Osteoarthritis

doi: 10.1101/2025.03.18.643878

Figure Lengend Snippet: (A) Clustering analysis was performed on concatenated monocytes stimulated with M-CSF, IL1β/TNFɑ, LPS, sialidase, and SF1-7. Subsequently, monocytes were downsampled to 100,000 cells before performing clustering. This generated six monocyte subpopulations, the table displays each population and its count, respectively. (B) Heatmap analysis demonstrate the six populations and their relative expression of TLR4, CD16, Siglec-9, Siglec-5, and CD14. (C) Each UMAP contour density plot represents one condition, i.e. M-CSF, IL1β/TNFɑ, LPS, sialidase, and SF1-7. (D) The concentration of IL-6 (pg/mL) produced by the stimulated monocytes were quantified, grey bars represent SFs and black bars represent the pathway-specific stimulations. The mean concentration is observed in each bar (D). Data is represented as mean±SD. For the monocytes stimulated with SF1-5, three technical replicates were used from three biological replicates. For monocytes stimulated with M-CSF, IL1β/TNFɑ, LPS, sialidase, three technical replicates were used from one biological replicate.

Techniques: Generated, Expressing, Concentration Assay, Produced

$(A) The effect on monocytes imposed by blocking TLR4 with TAK-242 was measured through cytokine production, a panel of cytokines and chemokines were screened after incubation with M-CSF, IL1β/TNFɑ, LPS, and sialidase for 24hrs with and without TAK-242. Each dataset is the mean of three biological replicates with three technical replicates pooled together. (B) The percentage of intermediate monocytes was quantified with and without TAK-242 ( n =2-3 form three biological replicates). (C-E) MFI of TLR4, Siglec-5, and Siglec-9 was compared between the stimulation in absence or presence of TAK-242 ( n =2-3 form three biological replicates, except TLR4 which was measured in one biological replicate). Heatmap analysis display data as fractions where the 0 is the smallest value of each sub column and the largest is the sum of all values in the sub column. Cytokine levels are indicated by Normalized Expression Levels and the % Cytokines in Presence of TAK-242 indicates how effective TAK-242 was at reducing cytokine secretion compared to without TAK-242. Data is represented as mean±SD, statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparisons test (* p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001).$

Journal: bioRxiv

Article Title: Sialoglycans modulate Siglec-5 – TLR4 Interactions in Osteoarthritis

doi: 10.1101/2025.03.18.643878

Figure Lengend Snippet: (A) The effect on monocytes imposed by blocking TLR4 with TAK-242 was measured through cytokine production, a panel of cytokines and chemokines were screened after incubation with M-CSF, IL1β/TNFɑ, LPS, and sialidase for 24hrs with and without TAK-242. Each dataset is the mean of three biological replicates with three technical replicates pooled together. (B) The percentage of intermediate monocytes was quantified with and without TAK-242 ( n =2-3 form three biological replicates). (C-E) MFI of TLR4, Siglec-5, and Siglec-9 was compared between the stimulation in absence or presence of TAK-242 ( n =2-3 form three biological replicates, except TLR4 which was measured in one biological replicate). Heatmap analysis display data as fractions where the 0 is the smallest value of each sub column and the largest is the sum of all values in the sub column. Cytokine levels are indicated by Normalized Expression Levels and the % Cytokines in Presence of TAK-242 indicates how effective TAK-242 was at reducing cytokine secretion compared to without TAK-242. Data is represented as mean±SD, statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparisons test (* p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001).

Techniques: Blocking Assay, Incubation, Expressing

(A) Representative immunofluorescence images of monocytes stimulated with M-CSF, LPS, and sialidase. Scale bar represents 20µm. (B) Quantification of % colocalization of TLR4 and Siglec-5. Each data point represents one cell, 5-7 images were captured per condition, two biological replicates were used. Data is represented as mean±SD, statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test (* p <0.05).

Journal: bioRxiv

Article Title: Sialoglycans modulate Siglec-5 – TLR4 Interactions in Osteoarthritis

doi: 10.1101/2025.03.18.643878

Figure Lengend Snippet: (A) Representative immunofluorescence images of monocytes stimulated with M-CSF, LPS, and sialidase. Scale bar represents 20µm. (B) Quantification of % colocalization of TLR4 and Siglec-5. Each data point represents one cell, 5-7 images were captured per condition, two biological replicates were used. Data is represented as mean±SD, statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test (* p <0.05).

Techniques: Immunofluorescence

Antibodies and protocols for indirect immunofluorescence analysis.

Journal: Journal of Clinical Medicine

Article Title: Th2 Cytokines Affect the Innate Immune Barrier without Impairing the Physical Barrier in a 3D Model of Normal Human Skin

doi: 10.3390/jcm12051941

Figure Lengend Snippet: Antibodies and protocols for indirect immunofluorescence analysis.

Article Snippet: Monoclonal mouse anti-human TLR4 (Novus Bio) , , 1:300 1 h at 37 °C.

Techniques: Immunofluorescence, Incubation

TLR2, TLR4, TLR7, and TLR9 immunofluorescence analysis on paraffin human skin sections. Representative TLR2 ( A , B ), TLR4 ( C , D ), TLR7 ( E , F ), and TLR9 ( G , H ) immunostainings in normal human skin paraffin sections. ( A , C , E , G ): samples harvested at 24 h; ( B , D , F , H ): samples harvested at 48 h. ( A – H ): control samples. Nuclei are counterstained with DAPI. TLR2: Toll-like receptor 2; TLR4: Toll-like receptor 4; TLR7: Toll-like receptor 7; TLR9: Toll-like receptor 9; DAPI: 4′, 6-diamidino-2-phenylindoledihydrochloride. White dotted line indicates the basal membrane. White arrows indicate positive immunostaining—scale bars: 50 µm.

Journal: Journal of Clinical Medicine

Article Title: Th2 Cytokines Affect the Innate Immune Barrier without Impairing the Physical Barrier in a 3D Model of Normal Human Skin

doi: 10.3390/jcm12051941

Figure Lengend Snippet: TLR2, TLR4, TLR7, and TLR9 immunofluorescence analysis on paraffin human skin sections. Representative TLR2 ( A , B ), TLR4 ( C , D ), TLR7 ( E , F ), and TLR9 ( G , H ) immunostainings in normal human skin paraffin sections. ( A , C , E , G ): samples harvested at 24 h; ( B , D , F , H ): samples harvested at 48 h. ( A – H ): control samples. Nuclei are counterstained with DAPI. TLR2: Toll-like receptor 2; TLR4: Toll-like receptor 4; TLR7: Toll-like receptor 7; TLR9: Toll-like receptor 9; DAPI: 4′, 6-diamidino-2-phenylindoledihydrochloride. White dotted line indicates the basal membrane. White arrows indicate positive immunostaining—scale bars: 50 µm.

Article Snippet: Monoclonal mouse anti-human TLR4 (Novus Bio) , , 1:300 1 h at 37 °C.

Techniques: Immunofluorescence, Control, Membrane, Immunostaining

TLR2, TLR4, TLR7, and TLR9 immunofluorescence analysis on IL-4 and IL-13 incubated paraffin human skin sections. Representative TLR2 ( A – D ), TLR4 ( E – H ), TLR7 ( I – L ), and TLR9 ( M – P ) immunostainings in normal human skin paraffin sections. ( A , E , I , M , C , G , K , O ): samples harvested at 24 h; ( B , F , J , N , D , H , L , P ): samples harvested at 48 h. Nuclei are counterstained with DAPI. TLR2: Toll-like receptor 2; TLR4: Toll-like receptor 4; TLR7: Toll-like receptor 7; TLR9: Toll-like receptor 9; IL-4: interleukin 4; IL-13: interleukin 13; DAPI: 4′, 6-diamidino-2-phenylindoledihydrochloride. White dotted line indicates the basal membrane. White arrows indicate positive immunostaining—scale bars: 50 µm.

Journal: Journal of Clinical Medicine

Article Title: Th2 Cytokines Affect the Innate Immune Barrier without Impairing the Physical Barrier in a 3D Model of Normal Human Skin

doi: 10.3390/jcm12051941

Figure Lengend Snippet: TLR2, TLR4, TLR7, and TLR9 immunofluorescence analysis on IL-4 and IL-13 incubated paraffin human skin sections. Representative TLR2 ( A – D ), TLR4 ( E – H ), TLR7 ( I – L ), and TLR9 ( M – P ) immunostainings in normal human skin paraffin sections. ( A , E , I , M , C , G , K , O ): samples harvested at 24 h; ( B , F , J , N , D , H , L , P ): samples harvested at 48 h. Nuclei are counterstained with DAPI. TLR2: Toll-like receptor 2; TLR4: Toll-like receptor 4; TLR7: Toll-like receptor 7; TLR9: Toll-like receptor 9; IL-4: interleukin 4; IL-13: interleukin 13; DAPI: 4′, 6-diamidino-2-phenylindoledihydrochloride. White dotted line indicates the basal membrane. White arrows indicate positive immunostaining—scale bars: 50 µm.

Article Snippet: Monoclonal mouse anti-human TLR4 (Novus Bio) , , 1:300 1 h at 37 °C.

Techniques: Immunofluorescence, Incubation, Membrane, Immunostaining

TLR2, TLR4, TLR7, and TLR9 immunofluorescence analysis on IL-22 and IL-23 incubated paraffin human skin sections. Representative TLR2 ( A – D ), TLR4 ( E – H ), TLR7 ( I – L ), and TLR9 ( M – P ) immunostainings in normal human skin paraffin sections. ( A , E , I , M , C , G , K , O ): samples harvested at 24 h; ( B , F , J , N , D , H , L , P ): samples harvested at 48 h. Nuclei are counterstained with DAPI. TLR2: Toll-like receptor 2; TLR4: Toll-like receptor 4; TLR7: Toll-like receptor 7; TLR9: Toll-like receptor 9; IL-22: interleukin 22; IL-23: interleukin 23; DAPI: 4′,6-diamidino-2-phenylindoledihydrochloride. White dotted line indicates the basal membrane. White arrows indicate positive immunostaining—scale bars: 50 µm.

Journal: Journal of Clinical Medicine

Article Title: Th2 Cytokines Affect the Innate Immune Barrier without Impairing the Physical Barrier in a 3D Model of Normal Human Skin

doi: 10.3390/jcm12051941

Figure Lengend Snippet: TLR2, TLR4, TLR7, and TLR9 immunofluorescence analysis on IL-22 and IL-23 incubated paraffin human skin sections. Representative TLR2 ( A – D ), TLR4 ( E – H ), TLR7 ( I – L ), and TLR9 ( M – P ) immunostainings in normal human skin paraffin sections. ( A , E , I , M , C , G , K , O ): samples harvested at 24 h; ( B , F , J , N , D , H , L , P ): samples harvested at 48 h. Nuclei are counterstained with DAPI. TLR2: Toll-like receptor 2; TLR4: Toll-like receptor 4; TLR7: Toll-like receptor 7; TLR9: Toll-like receptor 9; IL-22: interleukin 22; IL-23: interleukin 23; DAPI: 4′,6-diamidino-2-phenylindoledihydrochloride. White dotted line indicates the basal membrane. White arrows indicate positive immunostaining—scale bars: 50 µm.

Article Snippet: Monoclonal mouse anti-human TLR4 (Novus Bio) , , 1:300 1 h at 37 °C.

Techniques: Immunofluorescence, Incubation, Membrane, Immunostaining

Quantitative analysis of TLR2 and TLR 4 epidermal distribution after immunofluorescence analysis after 24 and 48 h of cytokine incubation. ( A ): TLR2; ( B ): TLR4. TLR2: Toll-like receptor 2; TLR4: Toll-like receptor 4; IL-4: interleukin 4; IL-13: interleukin 13; IL-22: interleukin 22; IL-23: interleukin 23. Statistical analysis was performed by Prism 9.0.0 via Kruskal–Wallis non-parametric analysis of variance, followed by Dunn’s post-hoc multiple comparison test. Differences were considered statistically significant when p < 0.05. * p < 0.05; *** p < 0.01.

Journal: Journal of Clinical Medicine

Article Title: Th2 Cytokines Affect the Innate Immune Barrier without Impairing the Physical Barrier in a 3D Model of Normal Human Skin

doi: 10.3390/jcm12051941

Figure Lengend Snippet: Quantitative analysis of TLR2 and TLR 4 epidermal distribution after immunofluorescence analysis after 24 and 48 h of cytokine incubation. ( A ): TLR2; ( B ): TLR4. TLR2: Toll-like receptor 2; TLR4: Toll-like receptor 4; IL-4: interleukin 4; IL-13: interleukin 13; IL-22: interleukin 22; IL-23: interleukin 23. Statistical analysis was performed by Prism 9.0.0 via Kruskal–Wallis non-parametric analysis of variance, followed by Dunn’s post-hoc multiple comparison test. Differences were considered statistically significant when p < 0.05. * p < 0.05; *** p < 0.01.

Article Snippet: Monoclonal mouse anti-human TLR4 (Novus Bio) , , 1:300 1 h at 37 °C.

Techniques: Immunofluorescence, Incubation, Comparison

$(A) Washed human platelets were treated with 0.4 U/mL thrombin at 37°C for 20 min, and the level of TLR4 on the surface of platelets was determined by flow cytometry. (B) Washed human platelets were treated with 0.1–0.4 U/mL thrombin at 37°C for 20 min and analyzed by flow cytometry for the surface level of TLR4 (n = 3). (C) The total and membrane protein fraction were extracted, and the TLR4 levels were further confirmed by western blot analysis and detected with anti-TLR4 antibody. α-tubulin served as the loading control in this assay. The bar graph showed the quantification of western blot analysis using densitometry. (D) Human platelets were directly treated with SFLLRN, AYPGKF, or SFLLRN plus AYPGKF at 37°C for 20 min. The level of surface TLR4 was determined by flow cytometry. (E) Washed human platelets were pretreated with U73122 or Y27632 at 28°C for 60 min followed by 0.4 U/mL thrombin treatments at 37°C for 20 min, and the surface level of TLR4 on the platelets was determined by flow cytometry. The m-3M3FBS was pretreated at 28°C for 60 min, than incubate at at 37°C for 20 min. The data represented the results of 5 independent experiments (mean ± SD; * p <0.05).$

Journal: PLoS ONE

Article Title: The Role of Calpain-Myosin 9-Rab7b Pathway in Mediating the Expression of Toll-Like Receptor 4 in Platelets: A Novel Mechanism Involved in α-Granules Trafficking

doi: 10.1371/journal.pone.0085833

Figure Lengend Snippet: (A) Washed human platelets were treated with 0.4 U/mL thrombin at 37°C for 20 min, and the level of TLR4 on the surface of platelets was determined by flow cytometry. (B) Washed human platelets were treated with 0.1–0.4 U/mL thrombin at 37°C for 20 min and analyzed by flow cytometry for the surface level of TLR4 (n = 3). (C) The total and membrane protein fraction were extracted, and the TLR4 levels were further confirmed by western blot analysis and detected with anti-TLR4 antibody. α-tubulin served as the loading control in this assay. The bar graph showed the quantification of western blot analysis using densitometry. (D) Human platelets were directly treated with SFLLRN, AYPGKF, or SFLLRN plus AYPGKF at 37°C for 20 min. The level of surface TLR4 was determined by flow cytometry. (E) Washed human platelets were pretreated with U73122 or Y27632 at 28°C for 60 min followed by 0.4 U/mL thrombin treatments at 37°C for 20 min, and the surface level of TLR4 on the platelets was determined by flow cytometry. The m-3M3FBS was pretreated at 28°C for 60 min, than incubate at at 37°C for 20 min. The data represented the results of 5 independent experiments (mean ± SD; * p <0.05).

Article Snippet: The beads containing immunoprecipitates were then resuspended in 2× SDS-PAGE sample buffer, and the reactions were subjected to western detection and analysis with mouse monoclonal anti-human TLR4 antibody (clone: 76B357.1; Abcam, Cambridge, MA, USA) or rabbit polyclonal anti-myosin-9 antibody (catalog no: GTX13236; GeneTex Co., Irvine, CA, USA).

Techniques: Flow Cytometry, Western Blot

(A) Human platelets were treated with thrombin at 37°C for 1 minute (black) or 20 minutes (gray), and the residual levels of calpain activity were determined. The data represented the results of 5 independent experiments (mean ± SD; * p <0.05). (B) Human platelets were pretreated with calpeptin at 28°C for 60 min followed by thrombin treatment at 37°C for 20 minutes (upper) or 3 mM CaCl 2 treatment at 37°C for 20–30 minutes (bottom). The total protein was extracted, and the calpain levels were analyzed by western blot and detected with the anti-calpain antibody. β-actin protein served as the loading control. (C) Human platelets were pretreated with calpeptin or TMB-8 at 28°C for 60 min followed by thrombin treatment at 37°C for 20 minutes. The platelet surface TLR4 level was determined by flow cytometry. The data represented the results of 5 independent experiments (mean ± SD; * p <0.05). (D) Human platelets were pretreated with calpeptin or TMB-8 at 28°C for 60 min followed by thrombin treatment at 37°C for 20 minutes (upper) or 3 mM CaCl 2 treatment at 37°C for 20–30 minutes (bottom). The membrane proteins were extracted, and the TLR4 level was further confirmed by western blot. α-tubulin protein served as the loading control. The bar graph showed the quantification of western blot analysis using densitometry. (E) Human platelets were directly treated with SFLLRN, AYPGKF, or m-3M3FBS or pretreated with U73122 at 28°C for 60 min followed by thrombin treatment at 37°C for 20 min, and the residual levels of calpain activity were determined. The data represented the results of 5 independent experiments (mean ± SD; * p <0.05).

Journal: PLoS ONE

Article Title: The Role of Calpain-Myosin 9-Rab7b Pathway in Mediating the Expression of Toll-Like Receptor 4 in Platelets: A Novel Mechanism Involved in α-Granules Trafficking

doi: 10.1371/journal.pone.0085833

Figure Lengend Snippet: (A) Human platelets were treated with thrombin at 37°C for 1 minute (black) or 20 minutes (gray), and the residual levels of calpain activity were determined. The data represented the results of 5 independent experiments (mean ± SD; * p <0.05). (B) Human platelets were pretreated with calpeptin at 28°C for 60 min followed by thrombin treatment at 37°C for 20 minutes (upper) or 3 mM CaCl 2 treatment at 37°C for 20–30 minutes (bottom). The total protein was extracted, and the calpain levels were analyzed by western blot and detected with the anti-calpain antibody. β-actin protein served as the loading control. (C) Human platelets were pretreated with calpeptin or TMB-8 at 28°C for 60 min followed by thrombin treatment at 37°C for 20 minutes. The platelet surface TLR4 level was determined by flow cytometry. The data represented the results of 5 independent experiments (mean ± SD; * p <0.05). (D) Human platelets were pretreated with calpeptin or TMB-8 at 28°C for 60 min followed by thrombin treatment at 37°C for 20 minutes (upper) or 3 mM CaCl 2 treatment at 37°C for 20–30 minutes (bottom). The membrane proteins were extracted, and the TLR4 level was further confirmed by western blot. α-tubulin protein served as the loading control. The bar graph showed the quantification of western blot analysis using densitometry. (E) Human platelets were directly treated with SFLLRN, AYPGKF, or m-3M3FBS or pretreated with U73122 at 28°C for 60 min followed by thrombin treatment at 37°C for 20 min, and the residual levels of calpain activity were determined. The data represented the results of 5 independent experiments (mean ± SD; * p <0.05).

Techniques: Activity Assay, Western Blot, Flow Cytometry

(A) Identification of myosin-9 as a TLR4-interacting protein by co-IP and mass spectrometry. Washed platelet lysates were prepared for IP with mouse IgG- or anti-TLR4-conjugated agarose beads. The precipitated proteins were resolved by SDS-PAGE and revealed by Coomassie Blue staining. The stars indicated the protein bands that were pulled down with the anti-TLR4 antibody but not by mouse IgG. The stars indicated myosin-9 that was identified by nano-LC/MS/MS on an LCQ Deca XP Plus ion trap mass spectrometer.

Journal: PLoS ONE

Article Title: The Role of Calpain-Myosin 9-Rab7b Pathway in Mediating the Expression of Toll-Like Receptor 4 in Platelets: A Novel Mechanism Involved in α-Granules Trafficking

doi: 10.1371/journal.pone.0085833

Figure Lengend Snippet: (A) Identification of myosin-9 as a TLR4-interacting protein by co-IP and mass spectrometry. Washed platelet lysates were prepared for IP with mouse IgG- or anti-TLR4-conjugated agarose beads. The precipitated proteins were resolved by SDS-PAGE and revealed by Coomassie Blue staining. The stars indicated the protein bands that were pulled down with the anti-TLR4 antibody but not by mouse IgG. The stars indicated myosin-9 that was identified by nano-LC/MS/MS on an LCQ Deca XP Plus ion trap mass spectrometer.

Techniques: Co-Immunoprecipitation Assay, Mass Spectrometry, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy

Platelets were treated with 5 µg/mL calpeptin followed by 4 U/mL thrombin for 20 minutes or 3 mM of CaCl2 for 30 minutes. (A and C) The interaction of myosin-9 with TLR4 was analyzed using immunoprecipitation. The pre-immune controls IgG were used to confirm the specificities of the TLR4 and myosin-9 antibodies. (B) The total protein extracted from treated platelets was used for immunoprecipitation with anti-myosin-9 antibody and immunoblotting with anti-TLR4 antibody. Immunoblotting with anti-myosin-9 antibody was used as an IP control. (D) The total protein extracted from treated platelets was used for immunoprecipitation with anti-TLR4 antibody and immunoblotting with anti-myosin-9 antibody. Immunoblotting with anti-TLR4 antibody was used as an IP control. The band density was determined using densitometry and was shown in the graph on the right. The data represented the results of three independent experiments (mean ± SD; * p <0.05 was considered significant).

Journal: PLoS ONE

Article Title: The Role of Calpain-Myosin 9-Rab7b Pathway in Mediating the Expression of Toll-Like Receptor 4 in Platelets: A Novel Mechanism Involved in α-Granules Trafficking

doi: 10.1371/journal.pone.0085833

Figure Lengend Snippet: Platelets were treated with 5 µg/mL calpeptin followed by 4 U/mL thrombin for 20 minutes or 3 mM of CaCl2 for 30 minutes. (A and C) The interaction of myosin-9 with TLR4 was analyzed using immunoprecipitation. The pre-immune controls IgG were used to confirm the specificities of the TLR4 and myosin-9 antibodies. (B) The total protein extracted from treated platelets was used for immunoprecipitation with anti-myosin-9 antibody and immunoblotting with anti-TLR4 antibody. Immunoblotting with anti-myosin-9 antibody was used as an IP control. (D) The total protein extracted from treated platelets was used for immunoprecipitation with anti-TLR4 antibody and immunoblotting with anti-myosin-9 antibody. Immunoblotting with anti-TLR4 antibody was used as an IP control. The band density was determined using densitometry and was shown in the graph on the right. The data represented the results of three independent experiments (mean ± SD; * p <0.05 was considered significant).

Techniques: Immunoprecipitation, Western Blot

Human platelets were pretreated with or without calpeptin at 28°C for 60 min followed by thrombin treatment at 37°C for 20 minutes. (A) The total protein extracted from platelets was used for western blot analysis and immunoblotting with anti-myosin-9 antibody. The β-actin protein served as the loading control. (B) The morphology of platelets were observed using TEM. Control human washed platelets are showed in B-1 and B-2. The platelet with randomly distribution of α-granules (AG) containing immuno-gold conjugated TLR4 (white arrow). Platelets with thrombin treatment at 37°C for 20 minutes were showed in B-3 and B-4. Original magnification of B-1 and B-3 are 40000×, B-2 and B-4 are 100000×. (C) The total protein extracted from platelets was used for immunoprecipitation with anti-Rab7b antibody and immunoblotting with anti-myosin-9 antibody. A pre-immune control IgG was used to confirm the specificity of the Rab7b antibody. Immunoblotting with anti-Rab7b antibody was used as an IP control.

Journal: PLoS ONE

Article Title: The Role of Calpain-Myosin 9-Rab7b Pathway in Mediating the Expression of Toll-Like Receptor 4 in Platelets: A Novel Mechanism Involved in α-Granules Trafficking

doi: 10.1371/journal.pone.0085833

Figure Lengend Snippet: Human platelets were pretreated with or without calpeptin at 28°C for 60 min followed by thrombin treatment at 37°C for 20 minutes. (A) The total protein extracted from platelets was used for western blot analysis and immunoblotting with anti-myosin-9 antibody. The β-actin protein served as the loading control. (B) The morphology of platelets were observed using TEM. Control human washed platelets are showed in B-1 and B-2. The platelet with randomly distribution of α-granules (AG) containing immuno-gold conjugated TLR4 (white arrow). Platelets with thrombin treatment at 37°C for 20 minutes were showed in B-3 and B-4. Original magnification of B-1 and B-3 are 40000×, B-2 and B-4 are 100000×. (C) The total protein extracted from platelets was used for immunoprecipitation with anti-Rab7b antibody and immunoblotting with anti-myosin-9 antibody. A pre-immune control IgG was used to confirm the specificity of the Rab7b antibody. Immunoblotting with anti-Rab7b antibody was used as an IP control.

Techniques: Western Blot, Immunoprecipitation

Thrombin may pass through the PAR1 and PAR4 receptors to activate downstream effectors for the PLC pathway but not the Rho pathway. The PLC pathway further activates calpain via calcium mobilization, and cleavages myosin-9, which decreases the interaction between myosin-9 and TLR4. In the other hand, myosin-9 does not coordinate with Rab7b to negatively regulate TLR4 containing α-granules trafficking in thrombin treated platelets, and leads to the increasing of TLR4 performance in thrombin-stimulated human platelets.

Journal: PLoS ONE

Article Title: The Role of Calpain-Myosin 9-Rab7b Pathway in Mediating the Expression of Toll-Like Receptor 4 in Platelets: A Novel Mechanism Involved in α-Granules Trafficking

doi: 10.1371/journal.pone.0085833

Figure Lengend Snippet: Thrombin may pass through the PAR1 and PAR4 receptors to activate downstream effectors for the PLC pathway but not the Rho pathway. The PLC pathway further activates calpain via calcium mobilization, and cleavages myosin-9, which decreases the interaction between myosin-9 and TLR4. In the other hand, myosin-9 does not coordinate with Rab7b to negatively regulate TLR4 containing α-granules trafficking in thrombin treated platelets, and leads to the increasing of TLR4 performance in thrombin-stimulated human platelets.

Techniques:

Immunocytochemical staining of TLR4 in (a) nonpermeabilized keratocytes and (b) permeabilized keratocytes (mag ×200) No TLR4 was detected in LESCs (data not shown).

Journal: BioMed Research International

Article Title: The Use of an IL-1 Receptor Antagonist Peptide to Control Inflammation in the Treatment of Corneal Limbal Epithelial Stem Cell Deficiency

doi: 10.1155/2015/516318

Figure Lengend Snippet: Immunocytochemical staining of TLR4 in (a) nonpermeabilized keratocytes and (b) permeabilized keratocytes (mag ×200) No TLR4 was detected in LESCs (data not shown).

Article Snippet: Cell were incubated in mouse anti-human TLR4 monoclonal antibody (AbCam, UK) diluted 1 : 50 in PGAS (0.2% gelatin from cold water fish skin, 0.02% saponin in PBS) for 1 hour in a humidified chamber followed by incubation with FITC conjugated goat anti-mouse IgG secondary antibody (AbCam, UK) diluted 1 : 100 in PGAS for 30 minutes at room temperature.

Techniques: Staining

A and B: Immunostaining of THP-1 cells with and without HCMV stimulation for TLR4 showed marked upregulation of TLR4-positive staining. C–E: Quantitative gene expression analysis using real-time qPCR in TLR4, MD2, and CD14 reflected similar upregulation seen in A. All mRNAs were analyzed from the same preparation. All data are means±S.D. of experiments performed in triplicate. Reaction mixtures without reverse transcriptase served as controls for genomic DNA contamination in all cases (Data not shown).

Journal: PLoS ONE

Article Title: Human Cytomegalovirus Induces TLR4 Signaling Components in Monocytes Altering TIRAP, TRAM and Downstream Interferon-Beta and TNF-Alpha Expression

doi: 10.1371/journal.pone.0044500

Figure Lengend Snippet: A and B: Immunostaining of THP-1 cells with and without HCMV stimulation for TLR4 showed marked upregulation of TLR4-positive staining. C–E: Quantitative gene expression analysis using real-time qPCR in TLR4, MD2, and CD14 reflected similar upregulation seen in A. All mRNAs were analyzed from the same preparation. All data are means±S.D. of experiments performed in triplicate. Reaction mixtures without reverse transcriptase served as controls for genomic DNA contamination in all cases (Data not shown).

Article Snippet: After fixation, the cells were permeabilized with 0.2% Triton X-100 (Sigma, St. Louis, MO) for 5 min at RT, blocked with normal donkey serum for 30 min and then incubated with primary mouse monoclonal anti-human TLR4 antibody in a moist chamber for 2 hrs at RT.

Techniques: Immunostaining, Staining, Expressing

All data are means±S.D. of experiments performed in triplicate. A: TIRAP levels were seen to increase even at the earliest times after incubation with HCMV. B: TRAM conversely showed a marked reduction of mRNA levels in response to HCMV, particularly at 6 hours. C: TRAF6, which potentially serves as a rate-limiting factor, was not found to be significantly altered at any time point. D and E: TLR4 regulating cytokine IL-6 and IL-8 expressions were elevated at 1 hour and 6 hours, respectively. F and G: The production of IL-6 and IL-8 measured by ELISA correlated well with the results of D and E. * p <0.05; ** p<0.01 by student T-test.

Journal: PLoS ONE

Article Title: Human Cytomegalovirus Induces TLR4 Signaling Components in Monocytes Altering TIRAP, TRAM and Downstream Interferon-Beta and TNF-Alpha Expression

doi: 10.1371/journal.pone.0044500

Figure Lengend Snippet: All data are means±S.D. of experiments performed in triplicate. A: TIRAP levels were seen to increase even at the earliest times after incubation with HCMV. B: TRAM conversely showed a marked reduction of mRNA levels in response to HCMV, particularly at 6 hours. C: TRAF6, which potentially serves as a rate-limiting factor, was not found to be significantly altered at any time point. D and E: TLR4 regulating cytokine IL-6 and IL-8 expressions were elevated at 1 hour and 6 hours, respectively. F and G: The production of IL-6 and IL-8 measured by ELISA correlated well with the results of D and E. * p <0.05; ** p<0.01 by student T-test.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

A: The increase in CD14 mRNA levels upon incubation with HCMV for 6 hours was significantly reduced by the TLR4 neutralizing antibody. B–F: Similar results were obtained for MD2 (B), TIRAP (C), IL-6 (E) and IL-8 (F). However, no effect on TRAM (D) was seen. (means±S.D. n = 4) (* p <0.05, **p<0.01 by students T test compared with control at 1 hour and 6 hours). G and H: ELISA analysis confirmed expected decreased levels of IL-6 (G) and IL-8 (H). All data are means of experiments performed in triplicate except where noted.

Journal: PLoS ONE

Article Title: Human Cytomegalovirus Induces TLR4 Signaling Components in Monocytes Altering TIRAP, TRAM and Downstream Interferon-Beta and TNF-Alpha Expression

doi: 10.1371/journal.pone.0044500

Figure Lengend Snippet: A: The increase in CD14 mRNA levels upon incubation with HCMV for 6 hours was significantly reduced by the TLR4 neutralizing antibody. B–F: Similar results were obtained for MD2 (B), TIRAP (C), IL-6 (E) and IL-8 (F). However, no effect on TRAM (D) was seen. (means±S.D. n = 4) (* p <0.05, **p<0.01 by students T test compared with control at 1 hour and 6 hours). G and H: ELISA analysis confirmed expected decreased levels of IL-6 (G) and IL-8 (H). All data are means of experiments performed in triplicate except where noted.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

CD14 mRNA (A) increases at 6 hours were blunted in the presence of TLR4/MD2 soluble receptors but not in media absent the exogenous soluble receptors, consistent with the results seen for neutralizing TLR4 antibody in . Similar effects were seen on TIRAP (B), IL-6 (E) and IL-8 (F), suggesting that TLR4/MD2 complex signaling was essential for IL-6 and IL-8 induction. However, the soluble receptors had a minimal effect on TRAM (C) and TRAF6 (D). (means±S.D. n = 4). IL-6 (G) and IL-8 (H) secretion measured by ELISA showed similar findings. All data are means + S.D. of experiments performed in triplicate unless otherwise noted. * p <0.05; ** p<0.01 by student T-test.

Journal: PLoS ONE

Article Title: Human Cytomegalovirus Induces TLR4 Signaling Components in Monocytes Altering TIRAP, TRAM and Downstream Interferon-Beta and TNF-Alpha Expression

doi: 10.1371/journal.pone.0044500

Figure Lengend Snippet: CD14 mRNA (A) increases at 6 hours were blunted in the presence of TLR4/MD2 soluble receptors but not in media absent the exogenous soluble receptors, consistent with the results seen for neutralizing TLR4 antibody in . Similar effects were seen on TIRAP (B), IL-6 (E) and IL-8 (F), suggesting that TLR4/MD2 complex signaling was essential for IL-6 and IL-8 induction. However, the soluble receptors had a minimal effect on TRAM (C) and TRAF6 (D). (means±S.D. n = 4). IL-6 (G) and IL-8 (H) secretion measured by ELISA showed similar findings. All data are means + S.D. of experiments performed in triplicate unless otherwise noted. * p <0.05; ** p<0.01 by student T-test.

Techniques: Enzyme-linked Immunosorbent Assay

TLR4 (A) and TIRAP (C) mRNA elevations at 6 hours of HCMV co-incubation were suppressed in the CD14 antisense-treated THP-1 cells, but little effect was seen on TRAM (D) and MD2 (B). However, IL-8 (F) and IFN-β (H) were abolished by the CD14 antisense whereas IL-6 (E) and TRIF (G) were unaffected, suggesting that CD14 is the responsible receptor for inducing IL-8 and IFN-β transcription via the TLR4/TIRAP-dependent pathway and possibly the TLR9/MyD88-dependent pathway, respectively. (means±S.D. n = 4) (* p <0.05, **p<0.01 by Student T-test compared with control at 1 hour and 6 hours) open squares = missense, closed squares = CD14 antisense. ELISA analysis showed that the CD14 blockade reduced the in vitro production of IL-6 (I), IL-8 (J) and sCD14 (K). Western blot for CD14 confirmed sequence-specific effect of the antisense (L). (n = 3). All data are means of experiments performed in triplicate except where noted.

Journal: PLoS ONE

Article Title: Human Cytomegalovirus Induces TLR4 Signaling Components in Monocytes Altering TIRAP, TRAM and Downstream Interferon-Beta and TNF-Alpha Expression

doi: 10.1371/journal.pone.0044500

Figure Lengend Snippet: TLR4 (A) and TIRAP (C) mRNA elevations at 6 hours of HCMV co-incubation were suppressed in the CD14 antisense-treated THP-1 cells, but little effect was seen on TRAM (D) and MD2 (B). However, IL-8 (F) and IFN-β (H) were abolished by the CD14 antisense whereas IL-6 (E) and TRIF (G) were unaffected, suggesting that CD14 is the responsible receptor for inducing IL-8 and IFN-β transcription via the TLR4/TIRAP-dependent pathway and possibly the TLR9/MyD88-dependent pathway, respectively. (means±S.D. n = 4) (* p <0.05, **p<0.01 by Student T-test compared with control at 1 hour and 6 hours) open squares = missense, closed squares = CD14 antisense. ELISA analysis showed that the CD14 blockade reduced the in vitro production of IL-6 (I), IL-8 (J) and sCD14 (K). Western blot for CD14 confirmed sequence-specific effect of the antisense (L). (n = 3). All data are means of experiments performed in triplicate except where noted.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, In Vitro, Western Blot, Sequencing

No change was seen for TLR4 (A) and TRAM (D) in the presence of IL-6 antibody. However, the IL-6 antibody had an inhibitory effect on CD14 (B), TIRAP (C), TRAF6 (E) and IL-8 (F). (means±S.D. n = 4) (* p <0.05, **p<0.01 by student T-test compared with control at 6 hours). The secretion of cytokines IL-8 (G) and sCD14 (H) from IL-6-antibody-treated THP-1 cells was analyzed by ELISA and the results were similar to those observed by real-time PCR. Elisa data are means of experiments performed in triplicate.

Journal: PLoS ONE

Article Title: Human Cytomegalovirus Induces TLR4 Signaling Components in Monocytes Altering TIRAP, TRAM and Downstream Interferon-Beta and TNF-Alpha Expression

doi: 10.1371/journal.pone.0044500

Figure Lengend Snippet: No change was seen for TLR4 (A) and TRAM (D) in the presence of IL-6 antibody. However, the IL-6 antibody had an inhibitory effect on CD14 (B), TIRAP (C), TRAF6 (E) and IL-8 (F). (means±S.D. n = 4) (* p <0.05, **p<0.01 by student T-test compared with control at 6 hours). The secretion of cytokines IL-8 (G) and sCD14 (H) from IL-6-antibody-treated THP-1 cells was analyzed by ELISA and the results were similar to those observed by real-time PCR. Elisa data are means of experiments performed in triplicate.

Techniques: Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Human Cytomegalovirus Induces TLR4 Signaling Components in Monocytes Altering TIRAP, TRAM and Downstream Interferon-Beta and TNF-Alpha Expression

doi: 10.1371/journal.pone.0044500

Figure Lengend Snippet: PCR Primer Sequences.

Techniques:

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