recombinant human il 1r1  (R&D Systems)

Journal: Cell reports

Article Title: NEMO recruitment at single cytokine-receptor complexes shows quantized dynamics independent of ligand affinity

doi: 10.1016/j.celrep.2025.116637

Figure Lengend Snippet: (A) Average distance trees of IL-1β full sequences across divergent species. (B) Average distance trees across predicted receptor-ligand binding regions. The red font indicates species selected for subsequent analysis. (C) Crystal and generated structure for IL-1β (blue) in complex with the extracellular domain of IL-1R1 (orange). (D) Differences in binding conformations across generated structures of IL-1β-IL-1R1 complexes for two predicted binding interfaces, with residues colored by their estimated contributions to the change in free energy. See also .

Article Snippet: Purified, carrier-free recombinant human IL-1R1 (R&D Systems) and IL-1β orthologues (Invitrogen, R&D Systems) were resuspended in 50% (v/v) glycerol in distilled H2O for a final glycerol concentration of 12%.

Techniques: Ligand Binding Assay, Generated, Binding Assay

Journal: Cell reports

Article Title: NEMO recruitment at single cytokine-receptor complexes shows quantized dynamics independent of ligand affinity

doi: 10.1016/j.celrep.2025.116637

Figure Lengend Snippet: (A) Schematic of IL-1β-induced EGFP-NEMO complex formation at the plasma membrane. IL-1β first binds to IL-1R1, enabling recruitment of IL-1R3 to form the receptor complex. Cytoplasmic MyD88 associates with the complex, facilitating IKK recruitment and the formation of EGFP-NEMO puncta. (B) Thermal shift curves of IL-1R1 stabilized by IL-1β indicate thermal stabilization of the human receptor by indicated cytokine orthologs. (C) Melting temperatures of isolated IL-1R1 as well as IL-1R1 in complex with IL-1β orthologs, derived from thermal shift curves in (B). (D) Quantitative descriptors extracted from each single-cell time courses of EGFP-NEMO puncta. (E) Boxplots of Fano noise evaluated for single-cell time courses at each concentration for indicated species. (F) Sigmoid curves fitted to the mean values of experimental single-cell descriptors across IL-1β concentrations and species. The EC 50 is indicated. See for fit parameters. (G) EC 50 values, reflecting the concentration at which 50% of the maximal response is reached quantified from (F). (H) Stochastic simulations using a minimal model recapitulate experimental results. Simulated dose-response curves as in (D) reveal dose-response relationshipsfor each predicted affinity. See for simulation and fit parameters. (I) EC 50 values derived from simulated data, reflecting the predicted net affinity of IL-1β to form signaling-competent complexes. See for fit parameters. See also .

Article Snippet: Purified, carrier-free recombinant human IL-1R1 (R&D Systems) and IL-1β orthologues (Invitrogen, R&D Systems) were resuspended in 50% (v/v) glycerol in distilled H2O for a final glycerol concentration of 12%.

Techniques: Clinical Proteomics, Membrane, Isolation, Derivative Assay, Concentration Assay

Journal: PLOS ONE

Article Title: Tannic acid, an IL-1β-direct binding compound, ameliorates IL-1β-induced inflammation and cartilage degradation by hindering IL-1β-IL-1R1 interaction

doi: 10.1371/journal.pone.0281834

Figure Lengend Snippet: (A) 2303 natural compound libraries were tested for their ability to inhibit the interaction between recombinant human IL-1β and IL1 Receptor 1. Microtiter 96-well plates were coated with hIL-1β (100 ng/well) overnight and the blocking buffer without any single compound was used as a positive control. Diluted single compounds (20 μM) were added to each well and incubated for 2 h. After washing, recombinant human IL-1R1 (125 ng/ml) was added and incubated for 2 h. Subsequently, HRP-conjugated Anti-Human IgG Fc (1:2000) was added and incubated for 1 h. An OD450 was obtained following the TMB reaction. Data indicate mean ± SD (n = 3). (B) Dose-dependency test of selected natural compound for blocking the interaction between recombinant human IL-1β and IL-1R1. Every step was identical to primary screening except for concentrations of selected natural compound (10, 40, or 160 μM) and washing buffer (PBS containing 0.05% Tween-20 and 0.01% Triton X-100). * p <0.05 compared to the control group of IL-1β and IL1 Receptor 1 interaction without any natural compounds. (C) IL-1β-dependent HEK-Blue IL-1β cells (5×10 4 cell/well) were seeded onto a 96-well plate and treated with pre-incubation (20 min) of human IL-1β (10 ng/ml) with various concentrations of TA (0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, or 100 μM). After a 24 h incubation, SEAP activity was assessed using QUANTI-Blue™ and the optical ensity (OD) at 620 nm. Cell viability was measured by analyzing the OD at 450 nm using D-Plus CCK. The IC 50 value of tannic acid was determined using the GraphPadPrism 10 software. Data indicate mean ± SD (n = 3). (D) Surface plasmon resonance (SPR) assay was used to analyze the direct binding of tannic acid to human IL-1β. Human IL-1β protein (50 μg/ml) was immobilized on a CM5 sensor chip and various concentrations of TA (1.56, 3.125, 6.25, 12.5, 25, 37.5, 50, 62.5, 75, 87.5, or 100 μΜ) were injected into the flow system with a flow rate 20 μl/min for 300 s and allowed to dissociate for 600 s. The K D values of the tannic acid against human IL-1β were obtained using the T200 BIA evaluation software.

Article Snippet: After three washes with PBST, recombinant human IL-1R1 protein (125 ng/ml, Abcam, Cambridge, UK) was added and incubated for 2 h. Subsequently, horseradish peroxidase (HRP)-conjugated anti-human IgG Fc (1:2000, Bethyl Laboratories, Montgomery, TX, USA) was added and incubated for 1 h. After washing, the TMB solution (Surmodics, Inc., Eden Prairie, MN, USA) was added to each well and incubated.

Techniques: Recombinant, Blocking Assay, Positive Control, Incubation, Activity Assay, Software, SPR Assay, Binding Assay, Injection

Journal: PLOS ONE

Article Title: Tannic acid, an IL-1β-direct binding compound, ameliorates IL-1β-induced inflammation and cartilage degradation by hindering IL-1β-IL-1R1 interaction

doi: 10.1371/journal.pone.0281834

Figure Lengend Snippet: Human articular chondrocytes from OA patients were seeded onto 12-well plates (2×10 5 cells/well) and serum-starved cells were co-treated with various concentrations of TA (0.5, 1, or 2 μM) or IL-1R1 (1 μg/ml) and IL-1β (10 ng/ml) for 48 h. (A) The mRNA expression levels of iNOS , COX-2 , IL-6 , and TNF were measured using qRT-PCR. The relative quantity of each gene expression was normalized to the relative quantity of human GADPH. (B) Griess reaction was used to measure the NO levels in the culture supernatants and PGE2, IL-6, and TNF levels in the culture supernatants were evaluated using ELISA. # p < 0.05 compared with medium only control group and * p <0.05 compared with IL-1β-treated group.

Article Snippet: After three washes with PBST, recombinant human IL-1R1 protein (125 ng/ml, Abcam, Cambridge, UK) was added and incubated for 2 h. Subsequently, horseradish peroxidase (HRP)-conjugated anti-human IgG Fc (1:2000, Bethyl Laboratories, Montgomery, TX, USA) was added and incubated for 1 h. After washing, the TMB solution (Surmodics, Inc., Eden Prairie, MN, USA) was added to each well and incubated.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: PLOS ONE

Article Title: Tannic acid, an IL-1β-direct binding compound, ameliorates IL-1β-induced inflammation and cartilage degradation by hindering IL-1β-IL-1R1 interaction

doi: 10.1371/journal.pone.0281834

Figure Lengend Snippet: Human articular chondrocytes from OA patients were seeded onto 6-well plates (3×10 5 cells/well) and serum-starved cells were co-treated with various concentrations of TA (2 μM), IL-1R1 (1 μg/ml), or anti-IL-1 neutralizing antibody (1 μg/ml) and IL-1β (10 ng/ml) for 48 h. mRNA and protein expressions of MMPs, ADAMTSs, collagen type II, and aggrecan were detected by qRT-PCR (A) and Western blot analysis (B). The relative quantity of each gene expression was normalized to the relative quantity of human GADPH and β-actin was used as a loading control. # p < 0.05 compared with medium only control group and * p <0.05 compared with IL-1β-treated group.

Article Snippet: After three washes with PBST, recombinant human IL-1R1 protein (125 ng/ml, Abcam, Cambridge, UK) was added and incubated for 2 h. Subsequently, horseradish peroxidase (HRP)-conjugated anti-human IgG Fc (1:2000, Bethyl Laboratories, Montgomery, TX, USA) was added and incubated for 1 h. After washing, the TMB solution (Surmodics, Inc., Eden Prairie, MN, USA) was added to each well and incubated.

Techniques: Quantitative RT-PCR, Western Blot, Expressing