recombinant mouse tlr4  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: Extracellular caspase-1: a critical inducer and a therapeutic target of lung injury in gut ischemia-reperfusion

doi: 10.3389/fimmu.2026.1811868

Figure Lengend Snippet: eCasp-1 binds to TLR4 to drive inflammation, which is effectively suppressed by the novel peptide C16. (A, B) Computational modeling predicted a strong interaction between eCasp-1 (red) and TLR4 (blue). (C) SPR analysis on the binding of eCasp-1 to TLR4 in vitro . (D, E) WT and TLR4 -/- peritoneal macrophages were treated with PBS or eCasp-1 (0.1 µg/ml) for 4 h. (D) IL-6 and (E) TNFα levels in the supernatants were measured by ELISA. (F, G) WT and TLR4 -/- mice received i.p. injections of PBS or eCasp-1 (5 µg/g BW), and plasma was collected 24 h later to measure (F) IL-6 and (G) TNFα. (H) In-silico analysis identified a putative binding site for mouse eCasp-1 (red) on the extracellular domain of TLR4 (blue). (I) C16 (silver), a 16-amino-acid peptide mimic, was designed based on the predicted binding interface and exhibited strong binding to eCasp-1 (red). (J) Computational modeling predicted a potential interaction between the eCasp-1-C16 complex and TLR4. (K) SPR analysis of eCasp-1 binding to TLR4 in the presence or absence of C16. (L) WT peritoneal macrophages were treated with eCasp-1 (0.1 µg/mL) with increasing concentrations of C16 (0.1, 1, 10 µg/mL) for 4 h, and TNFα levels in the supernatants were measured by ELISA. Experiments were repeated 2–3 times and all the data obtained were used for analysis. Data were expressed as mean ± SEM (n = 5–9 samples/group) and compared by one-way analysis of variance and Student-Newman-Keuls method ( * p < 0.05 vs. WT PBS; # p < 0.05 vs. WT with eCasp-1, (+)eCasp-1 (-)C16).

Article Snippet: Recombinant mouse TLR4 (rmTLR4; ≥90% purity) was purchased from R&D Systems (Cat. No. 9149-TR-050, Minneapolis, MN), supplied in carrier-free form, and reconstituted in sterile PBS according to the manufacturer’s instructions.

Techniques: Binding Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, In Silico

Journal: Frontiers in Immunology

Article Title: Extracellular caspase-1: a critical inducer and a therapeutic target of lung injury in gut ischemia-reperfusion

doi: 10.3389/fimmu.2026.1811868

Figure Lengend Snippet: Summary of findings. In gut I/R injury, inflammasomes activation promotes the cleavage of caspase-1 and the extracellular release of its p20 subunit through GSDMD-dependent membrane processes. This release may occur in association with GSDMD pore formation as well as membrane disruption during lytic cell death (pyroptosis). Once released, extracellular caspase-1 (eCasp-1) acts as a potent DAMP by binding to TLR4, thereby amplifying release of inflammatory cytokines, aggravating lung injury. Therapeutic intervention with the inhibitory peptide C16, which specifically blocks the eCasp-1-TLR4 interaction, effectively attenuates systemic inflammation and improves survival outcomes. I/R, Ischemia-reperfusion; GSDMD, Gasdermin-D; eCasp-1, Extracellular caspase-1; DAMP, Damage-associated molecular pattern; TLR4, Toll-like receptor 4.

Article Snippet: Recombinant mouse TLR4 (rmTLR4; ≥90% purity) was purchased from R&D Systems (Cat. No. 9149-TR-050, Minneapolis, MN), supplied in carrier-free form, and reconstituted in sterile PBS according to the manufacturer’s instructions.

Techniques: Activation Assay, Membrane, Disruption, Binding Assay

Journal: Communications Biology

Article Title: Adjuvant activity of a small molecule TLR4 agonist discovered via structure-based virtual screening

doi: 10.1038/s42003-025-08582-y

Figure Lengend Snippet: A The 2D structures of small molecule TLR4-binding compounds were visualised in PDB format using PyMOL Green indicates carbon (C), Red indicates oxygen (O), yellow-sulphur(S), and blue indicates Nitrogen. B Molecular docking of the compounds with mouse TLR4-MD2 receptors. The small molecule compounds (green) as well as MPLA (green) were docked at the active sites of the mouse TLR4-MD2 which includes LRR domain (shown in pink) and MD2 (shown in orange). C Ligplot analysis illustrating the interactions between the compounds and mouse TLR4-MD2 complex. Hydrophobic interactions are represented by red arcs, while hydrogen bonds are depicted as green dashed lines with bond lengths specified. D Molecular dynamics simulation analysis of compounds-mTLR4 complexes. A Root Mean Square Deviation (RMSD) of the protein backbone, indicating the stability of the protein-ligand complexes over simulation time b) Root Mean Square Fluctuation (RMSF), showing the flexibility of individual amino acid residues in the mTLR4 protein for each compound-TLR4 complex.

Article Snippet: Briefly, recombinant mouse TLR4 protein (R&D Systems, Minneapolis, MN) was fluorescently labelled using the Monolith NTTM Protein Labelling Kit RED-NHS (NanoTemper Technologies, Germany) according to the manufacturer’s protocol (NanoTemper Technologies, 2021).

Techniques: Binding Assay

Journal: Communications Biology

Article Title: Adjuvant activity of a small molecule TLR4 agonist discovered via structure-based virtual screening

doi: 10.1038/s42003-025-08582-y

Figure Lengend Snippet: A TLR4 agonist activity of compounds . WT and TLR4 −/− mouse macrophage cell lines were stimulated with 2 µg/mL MPLA (1.13 µM), or 2 µM of NSF-418 (0.679 µg/mL) or NSF-501(0.649 µg/mL) or NSF-951 (0.873 µg/mL) for 24hrs at 37 °C/5%CO 2 and production of proinflammatory cytokines (IL-6 and TNF-α) was analysed in the culture supernatant by sandwich ELISA as described in methodology. B TLR4 antagonist activity of compounds . RAW 264.7 cells were treated with the compounds as described above, followed by treatment with MPLA (2 µg/mL) for 24 hrs at 37 °C/ 5%CO 2 and production of proinflammatory cytokines (IL-6 and TNF-α) was analysed in the culture supernatant by sandwich ELISA as described in methodology. All data represent the mean ± SD of triplicates ( n = 3) and are representative of three independent biological experiments. Significant differences were analysed using one-way analysis of variance (ANOVA), followed by the Dunnett post hoc test. The value of p < 0.05 was considered statistically significant and noted as **** p < 0.0001 and “ns” as not significant.

Article Snippet: Briefly, recombinant mouse TLR4 protein (R&D Systems, Minneapolis, MN) was fluorescently labelled using the Monolith NTTM Protein Labelling Kit RED-NHS (NanoTemper Technologies, Germany) according to the manufacturer’s protocol (NanoTemper Technologies, 2021).

Techniques: Activity Assay, Sandwich ELISA

Journal: Nature Communications

Article Title: Gut microbiota-derived GlcNAc-MurNAc is a TLR4 agonist that protects the host gut

doi: 10.1038/s41467-025-60678-5

Figure Lengend Snippet: a SEAP reporter readouts from HEK-Blue TM NOD1/2 reporter cells stimulated with MDP (200 nM), M-AE (200 nM), GM (20 μM), or M (20 μM) for 18 h. Data are presented as mean values ( n = 3). b-g PGNs induce cytokine production in macrophages and monocytes and activate dendritic cells. The respective immune cells were stimulated with the indicated PGNs, followed by cytokine analysis by RT-qPCR ( c ) or ELISA ( d ). RAW264.7 cells were treated with GM (4 mM, 2 mM, 1 mM), M (4 mM), or MDP (0.4 mM) for 24 h ( n = 4). Murine bone marrow-derived macrophages (BMDMs) and THP-1 cells were treated with GM (8 mM, 4 mM, 2 mM), M (8 mM), or MDP (0.4 mM). n = 3 or 4, respectively. e Mean fluorescent intensity (MFI) of surface CD80/86 in the cDC2s population of murine bone marrow-derived dendritic cells (BMDMs) that were treated with GM (50, 100, and 200 μM) for 18 h. f-g Assays using polymyxin B (PB) confirmed that GM was not contaminated with trace endotoxin. f ELISA analysis of the TNF- α levels in BMDMs stimulated with GM (4 mM), LPS (100 ng/mL), or polymyxin B resin (PB-resin)-neutralized GM (4 mM) or LPS (100 ng/mL) for 24 h. g The TLR4 inhibitor specifically suppresses the GM-induced TNF- α production. The RAW_Dual TM cells were pre-incubated with the respective TLR inhibitors (10 μM) for 1 h followed by GM stimulation (2 mM) for 24 h. TLR2i, TLR4i, and TLR7/9i refer to CU_CPT22, TAK-242, and AT791, respectively. ( h ) SEAP reporter readouts from HEK-Blue TM mTLR4 reporter cells stimulated with GM (1 mM) or LPS (100 ng/mL) titrated with polymyxin B (0, 0.1, 1, 10 kU) for 24 h ( n = 4). Data are presented as mean values ± s.e.m. ( n = 3 unless otherwise noted) with indicated P values determined using one-way ANOVA followed by Dunnett’s multiple comparison for ( c-d, f-g ) or two-way ANOVA followed by Tukey’s multiple comparison for ( e, h ). All experiments were repeated twice with similar results. Source data are provided as a file.

Article Snippet: Recombinant mouse TLR4 (26-638)-His 6 protein (Cusabio, Cat. No. CSB-YP023603MO) was reconstituted in water and aliquoted for storage at −80 °C.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Derivative Assay, Incubation, Comparison

Journal: Nature Communications

Article Title: Gut microbiota-derived GlcNAc-MurNAc is a TLR4 agonist that protects the host gut

doi: 10.1038/s41467-025-60678-5

Figure Lengend Snippet: a-b WT and Tlr4 -/- RAW_Dual TM reporter cells were used to evaluate GM-induced NF-κB and IRF signaling. Cells were treated with respective PGNs at indicated concentrations for 24 h for quanti-blue analysis (top, n = 3) or quanti-luciferase analysis (bottom). LPS (100 ng/mL), MDP (0.4 mM), or Poly(I: C) (0.1 mg/mL) were used as controls. Concentrations of GM, MG, and M tested were indicated in the figures. c-e The RNAseq analysis and validation confirm that the GM-induced immune responses in BMDM require TLR4. c Heatmap plot of RNAseq results showing the mean values of differentially expressed genes ( p < 0.01, and logFC > 1) in WT and Tlr4 -/- BMDMs treated with GM (8 mM) or MDP (0.4 mM) for 24 h ( n = 3). d-e Validation of the RNAseq results by RT-qPCR ( d ) and ELISA ( e ). WT and Tlr4 -/- BMDMs were treated with GM (4 mM) or MDP (0.4 mM) for 24 h. Data are presented as mean values ± s.e.m. ( n = 4 unless otherwise noted) with indicated P values determined using one-way ANOVA followed by Dunnett’s multiple comparison for ( d-e ) or two-way ANOVA followed by Tukey’s multiple comparison for ( b ). All experiments were repeated twice with similar results. Source data are provided as a file.

Article Snippet: Recombinant mouse TLR4 (26-638)-His 6 protein (Cusabio, Cat. No. CSB-YP023603MO) was reconstituted in water and aliquoted for storage at −80 °C.

Techniques: Luciferase, Biomarker Discovery, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Nature Communications

Article Title: Gut microbiota-derived GlcNAc-MurNAc is a TLR4 agonist that protects the host gut

doi: 10.1038/s41467-025-60678-5

Figure Lengend Snippet: a-b In vitro enrichment of recombinant mTLR4 protein by immobilized GM. a Scheme of the pulldown assay, with GM- or G-immobilized agarose beads. b Coomassie blue-stained SDS-PAGE analysis of mTLR4 proteins bound or unbound to the resin. Excess GM was added to compete with mTLR4 bound to GM-immobilized beads. c Surface plasmon resonance (SPR) demonstrates direct binding of GM and mTLR4. Representative SPR sensorgrams of mTLR4 binding to MDP (left) and GM (middle) are shown. Corresponding plots and fitted data of steady-state binding for MDP (blue) and GM (black), with the estimated respective K D values, are shown (right). ND stands for not detected. The data shown are the representative results from 3 independent experiments. Source data are provided as a file.

Article Snippet: Recombinant mouse TLR4 (26-638)-His 6 protein (Cusabio, Cat. No. CSB-YP023603MO) was reconstituted in water and aliquoted for storage at −80 °C.

Techniques: In Vitro, Recombinant, Staining, SDS Page, SPR Assay, Binding Assay

Journal: Nature Communications

Article Title: Gut microbiota-derived GlcNAc-MurNAc is a TLR4 agonist that protects the host gut

doi: 10.1038/s41467-025-60678-5

Figure Lengend Snippet: a Analysis of specific residues in TLR4/MD-2 required for GM-induced NF-κB activity. The various mTLR4/mMD2 variants were transiently expressed in HEK-Blue TM reporter cells and treated with GM (0.4 mM, 0.2 mM) or LPS (100 ng/mL) for 24 h. The activation of NF-κB was measured based on the SEAP reporter signals. For each transfected group, the readouts were normalized to the H 2 O-treated samples. Data are presented as mean values ± s.e.m. ( n = 3). b-c Structural-activity relationship (SAR) analysis of disaccharide analogs using HEK-Blue TM mTLR4 reporter cells. Cells were treated with respective disaccharides at 0.5 or 1 mM for 24 h. LPS (20 ng/mL and 100 ng/mL) was used as controls. Data are presented as mean values ( n = 2). All experiments were repeated twice with similar results. Source data are provided as a file.

Article Snippet: Recombinant mouse TLR4 (26-638)-His 6 protein (Cusabio, Cat. No. CSB-YP023603MO) was reconstituted in water and aliquoted for storage at −80 °C.

Techniques: Activity Assay, Activation Assay, Transfection

Journal: Nature Communications

Article Title: Gut microbiota-derived GlcNAc-MurNAc is a TLR4 agonist that protects the host gut

doi: 10.1038/s41467-025-60678-5

Figure Lengend Snippet: a Timeline of the study, where GM was intraperitoneally administered at 10 mg/kg to mice (WT and Tlr4 -/- ) that were provided with 3% DSS or normal drinking water. I.p. injections of PBS were given to the negative control group. b-c Measurements of mice body weight ( b ) ( n = 8, 6, 10, 10, 9, 8, 10, 10, respectively) and colon length ( c ) ( n = 8, 6, 14, 14, 8, 8, 15, 14, respectively) at the endpoint of the timeline. d-e Representative images of colons ( d ) and H&E-stained colon sections ( e ) from each group ( n = 5). f Flow cytometry analysis of immune cell infiltration in the colons of each treatment group. The percentage of the CD45 + population ( n = 8, 6, 12, 13, 7, 8, 12, 12, respectively) was calculated within pre-gated live cells. The percentage of Ly6C + MHCII - ( n = 8, 6, 10, 10, 7, 8, 10, 8, respectively) and Ly6C + Ly6G + ( n = 8, 6, 8, 8, 7, 8, 11, 9, respectively) populations was calculated within the CD45 + population. g RT-qPCR analysis of colonic cytokines demonstrates the significantly reduced inflammation in the GM-supplemented DSS-induced colitis in WT mice but not Tlr4 -/- mice. Tnfα , n = 10, 10, 9, 9, 5, 8, 8, 8, respectively. Il1β , n = 12, 12, 10, 13, 5, 8, 8, 7, respectively. Ccl2 , n = 10, 10, 9, 9, 5, 8, 6, 6, respectively. Data are presented as mean values ± s.e.m. ( n as indicated) with indicated P values determined using two-way ANOVA followed by Tukey’s multiple comparison for ( c, f-g ). All experiments were repeated twice with similar results. Source data are provided as a file.

Article Snippet: Recombinant mouse TLR4 (26-638)-His 6 protein (Cusabio, Cat. No. CSB-YP023603MO) was reconstituted in water and aliquoted for storage at −80 °C.

Techniques: Negative Control, Staining, Flow Cytometry, Quantitative RT-PCR, Comparison