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: BMC Oral Health

Article Title: Edaravone attenuates doxorubicin-induced oral mucosal injury via modulation of oxidative stress, inflammatory signaling, and the SIRT1/TLR4/NF-kB/ACE2 axis in rats

doi: 10.1186/s12903-025-07148-y

Figure Lengend Snippet: Tongue tissue TLR4 and proinflammatory cytokines. A TLR4 (ng/mg protein), ( B ) TNF-α (ng/L/g), ( C ) IL-6 (ng/L/g) (mean ± SEM). DOX increased TLR4/TNF-α/IL-6; EDO reduced these levels. Statistics: TLR4/IL-6—ANOVA ± Tukey; TNF-α—Kruskal–Wallis ± Mann–Whitney. Significance: * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant (adjusted p ≥ 0.05)

Article Snippet: TLR4 protein levels in tongue tissue were quantified using a rat TLR4 ELISA kit (Cusabio, Cat. No: CSB-E15822r).

Techniques: MANN-WHITNEY

Journal: mBio

Article Title: Distinct immune properties of the N- and C-termini of the immunosuppressive domain of Ebola virus glycoprotein

doi: 10.1128/mbio.02278-25

Figure Lengend Snippet: TLR4 is involved in the capture of shed EBOV GP. ( A ) Binding and internalization assays on 293 cells stably expressing TLR4 (293-TLR4) and THP-1 cells were analyzed by western blotting. Cells were treated or mock-treated with recombinant TLR4 (rTLR4) and then cultured with medium or WT, mut 5, and mut 14 EBOV shed GP. Cells were then treated with trypsin to evaluate internalization of shed GP. Cell pellets were immunostained for TLR4, EBOV GP, and GAPDH as an internal control. ( B, C ) Induction of NFκB and NFAT. 293-TLR4 cells were transfected with NFAT-Luc ( B ) or NFκB-Luc ( C ), treated with CLI-095 or rTLR4 with or without CsA, treated or mock-treated with WT, mut 5, or mut 14 EBOV shed GP, and subjected to luciferase assays. Two-way ANOVA followed by a Tukey’s multiple comparison test: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: 293T and 293-TLR4 cells were seeded at 10 5 cells per well in 12-well plates (Sigma-Aldrich), transfected with NFκB-Luc (Addgene, #111216) or NFAT-Luc (Addgene, #17870) plasmids using TransIT LT1 transfection reagent (Mirus Bio LLC) and incubated at 37°C for 48 h. Cells were then stimulated with 25 ng/mL TPA and 0.5 μM of ionomycin, or 1 μM of CsA, 10 μg/mL of rTLR4 (RnD Systems, #1478-TR-050), or 100 ng/mL CLI-095 (InvivoGen) for 1 h. Next, cells were pulsed with medium alone or with EBOV VLPs for an additional 24 h. Then, cells were lysed with Pierce Luciferase Cell lysis buffer (Thermo Fisher Scientific), and cell lysates were assayed for luciferase activity using a luminometer (Glomax 20/20, Promega).

Techniques: Binding Assay, Stable Transfection, Expressing, Western Blot, Recombinant, Cell Culture, Control, Transfection, Luciferase, Comparison