mab anti human tlr4  (Santa Cruz Biotechnology)

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: The following antibodies were used: rabbit polyclonal anti-IFI16 N-term and C-term (produced as described in ), mAb anti-human TLR4 (sc-293072, Santa Cruz Biotechnologies), mAb anti-human TLR4 (sc-13593, Santa Cruz Biotechnologies), mAb anti-β-actin (A1978, Sigma-Aldrich), rabbit IgG-HRP (A6154, Sigma-Aldrich), and mouse IgG-HRP (A16072, Thermo Fisher Scientific). pET30a expression vectors encoding the human and murine proteins used in this study were purchased from GenScript.

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: The following antibodies were used: rabbit polyclonal anti-IFI16 N-term and C-term (produced as described in ), mAb anti-human TLR4 (sc-293072, Santa Cruz Biotechnologies), mAb anti-human TLR4 (sc-13593, Santa Cruz Biotechnologies), mAb anti-β-actin (A1978, Sigma-Aldrich), rabbit IgG-HRP (A6154, Sigma-Aldrich), and mouse IgG-HRP (A16072, Thermo Fisher Scientific). pET30a expression vectors encoding the human and murine proteins used in this study were purchased from GenScript.

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: The following antibodies were used: rabbit polyclonal anti-IFI16 N-term and C-term (produced as described in ), mAb anti-human TLR4 (sc-293072, Santa Cruz Biotechnologies), mAb anti-human TLR4 (sc-13593, Santa Cruz Biotechnologies), mAb anti-β-actin (A1978, Sigma-Aldrich), rabbit IgG-HRP (A6154, Sigma-Aldrich), and mouse IgG-HRP (A16072, Thermo Fisher Scientific). pET30a expression vectors encoding the human and murine proteins used in this study were purchased from GenScript.

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: The following antibodies were used: rabbit polyclonal anti-IFI16 N-term and C-term (produced as described in ), mAb anti-human TLR4 (sc-293072, Santa Cruz Biotechnologies), mAb anti-human TLR4 (sc-13593, Santa Cruz Biotechnologies), mAb anti-β-actin (A1978, Sigma-Aldrich), rabbit IgG-HRP (A6154, Sigma-Aldrich), and mouse IgG-HRP (A16072, Thermo Fisher Scientific). pET30a expression vectors encoding the human and murine proteins used in this study were purchased from GenScript.

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: The following antibodies were used: rabbit polyclonal anti-IFI16 N-term and C-term (produced as described in ), mAb anti-human TLR4 (sc-293072, Santa Cruz Biotechnologies), mAb anti-human TLR4 (sc-13593, Santa Cruz Biotechnologies), mAb anti-β-actin (A1978, Sigma-Aldrich), rabbit IgG-HRP (A6154, Sigma-Aldrich), and mouse IgG-HRP (A16072, Thermo Fisher Scientific). pET30a expression vectors encoding the human and murine proteins used in this study were purchased from GenScript.

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

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

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

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).

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:

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.

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: Generated, Expressing, Concentration Assay, Produced

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).

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: Blocking Assay, Incubation, Expressing

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).

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: Immunofluorescence