anti human tlr6 mab  (Hycult Biotech)

Journal: iScience

Article Title: Neuregulin-1/ErbB4 signaling modulates Plasmodium falciparum HRP2-induced damage to brain cortical organoids

doi: 10.1016/j.isci.2022.104407

Figure Lengend Snippet: HRP2 increases pro-inflammatory and Toll-like receptor (TLR) genes in cortical organoids. HRP2-induced inflammation-related genes were profiled Fold changes greater than 1.5 and p values < 0.05 calculated using Student’s t -test was considered to be a significant dysregulation. (A) A heatmap was generated showing up-and down-regulated genes. The effects of HRP2 on cortical organoids at day 50 in culture are shown by site (B), including cerebral cortex and glia, as well as by pathway (C), including TLR signaling. Further analysis revealed the relationship between upregulated genes and activation of TLR2 pathway (D). Western blot densitometry of organoid lysates (data represented as means ± SEM) confirms that HRP2 significantly induces TLR2 and TLR1 expression, whereas NRG1 reduces these effects (E, F). a: p < 0.05 compared to basal conditions; b: p < 0.05 compared to HRP2 treatment. One-way ANOVA and t -test were used. Please also see Figure S4 .

Article Snippet: Rabbit monoclonal anti-human TLR6 (clone D1Z88) , Cell Signaling , Cat #12717T RRID: AB_2798005.

Techniques: Generated, Activation Assay, Western Blot, Expressing

Journal: iScience

Article Title: Neuregulin-1/ErbB4 signaling modulates Plasmodium falciparum HRP2-induced damage to brain cortical organoids

doi: 10.1016/j.isci.2022.104407

Figure Lengend Snippet: HRP2 uses TLR1 and TLR2 to increase CXCL10 expression and cell death in iPSC Cell proliferation, apoptosis, and expression of inflammatory markers were assessed in iPSC treated with HRP2 after TLR1 and TLR2 blockage. (A) Proliferation assay showed that IPSC growth was partially restored, whereas their apoptosis (B) and necrosis (C) were reduced to the levels of untreated controls. TLR1 and TLR2 blocking in HRP2-treated iPSC has an antiapoptotic and anti-inflammatory effect, as it is confirmed by IHC: the expression of cleaved caspase 3 (D, F) and CXCL10 (E, G) is reduced. Data represented as means ± SEM a: p < 0.05 compared to basal conditions; b: p < 0.05 compared to HRP2 treatment. Statistical significance between groups was determined using t -test and analysis of variance.

Article Snippet: Rabbit monoclonal anti-human TLR6 (clone D1Z88) , Cell Signaling , Cat #12717T RRID: AB_2798005.

Techniques: Expressing, Proliferation Assay, Blocking Assay

Journal: iScience

Article Title: Neuregulin-1/ErbB4 signaling modulates Plasmodium falciparum HRP2-induced damage to brain cortical organoids

doi: 10.1016/j.isci.2022.104407

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-human TLR6 (clone D1Z88) , Cell Signaling , Cat #12717T RRID: AB_2798005.

Techniques: Functional Assay, Recombinant, Plasmid Preparation, CCK-8 Assay, Bicinchoninic Acid Protein Assay, Isolation, Expressing, Western Blot, Software, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex

doi: 10.1074/jbc.M113.453266

Figure Lengend Snippet: Recombinant sTLR proteins are monomeric, properly folded, and biologically functional. A, 100 μg of each purified sTLR protein as indicated was loaded for 7.5% SDS-PAGE and stained with Coomassie blue dye. B, each purified sTLR protein as indicated was analyzed by size exclusion chromatography using a Superdex 200 column. C, soluble TLRs were incubated in microtiter plate wells coated with either the anti-TLR1 mAb (clone GD2F4) or the anti-TLR2 mAb (T2.5) as indicated. Binding of soluble TLR1, TLR1P315L, or TLR2 was detected using HRP-conjugated anti-HA and anti-FLAG mAbs. D, SW620 cells were co-transfected with full-length TLR1, TLR2, an IL-8 promoter-driven luciferase reporter gene, and a Renilla luciferase transfection control. 48 h post-transfection, the cells were stimulated with 10 ng/ml Pam3CSK4 with or without 1 μg/ml soluble TLR1 or TLR2 as indicated. Firefly luciferase activities were normalized to that of the Renilla luciferase control. These values were normalized to that of empty CMV vector whose value was taken as 1. Error bars represent the S.D. of three independent events. mAu, milliabsorbance units.

Article Snippet: The unconjugated anti-human TLR1 mAb (clone GD2.F4, CD281) and anti-human TLR2 mAb (clone T2.5, CD282) were obtained from eBioscience (San Diego, CA).

Techniques: Recombinant, Functional Assay, Purification, SDS Page, Staining, Size-exclusion Chromatography, Incubation, Binding Assay, Transfection, Luciferase, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex

doi: 10.1074/jbc.M113.453266

Figure Lengend Snippet: LBP and soluble CD14 enhance soluble TLR1·TLR2·Pam3CSK4 ternary complex formation but are not part of the final ternary complex. In A–D, various combinations of 0.25 μm TLR1, 0.05 μm TLR2, 2.5 μm Pam3CSK4, 0.05 μm LBP, and/or 0.25 μm sCD14 were incubated for 2 h at 37 °C in a 500-μl volume of PBS buffer, pH 7.4 as indicated. Protein complexes were separated by size exclusion chromatography. The expected molecular weight of the TLR monomers and dimers was estimated by column calibration using known molecular weight standards. Proteins in eluted fractions were separated by 7.5% SDS-PAGE and transferred by Western blotting, and TLR1, TLR2, LBP, and sCD14 were detected using suitable antibodies and HRP conjugates (see “Experimental Procedures”). The results shown are representative of at least three independent experiments. mAu, milliabsorbance units.

Article Snippet: The unconjugated anti-human TLR1 mAb (clone GD2.F4, CD281) and anti-human TLR2 mAb (clone T2.5, CD282) were obtained from eBioscience (San Diego, CA).

Techniques: Incubation, Size-exclusion Chromatography, Molecular Weight, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex

doi: 10.1074/jbc.M113.453266

Figure Lengend Snippet: Soluble TLRs do not form homodimers when incubated with Pam3CSK4. 0.5 μm TLR1 (A), 0.5 μm TLR2 (B), a 0.25 μm concentration each of TLR1 and TLR2 (C), or a 0.25 μm concentration each of TLR1P315L and TLR2 (D) were preincubated with a 5-fold molar excess of Pam3CSK4 (2.5 μm) together with 0.05 μm LBP and 0.25 μm sCD14 in PBS, pH 7.4 buffer for 2 h at 37 °C in a 500-μl reaction volume. Proteins and protein complexes were separated and analyzed as described in the legend of Fig. 2. The results shown are representative of at least three independent experiments. mAu, milliabsorbance units.

Article Snippet: The unconjugated anti-human TLR1 mAb (clone GD2.F4, CD281) and anti-human TLR2 mAb (clone T2.5, CD282) were obtained from eBioscience (San Diego, CA).

Techniques: Incubation, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex

doi: 10.1074/jbc.M113.453266

Figure Lengend Snippet: LBP and soluble CD14 enhance complex formation between TLR1, TLR2, and the OspA lipoprotein of B. burgdorferi. In A–E, various combinations of 0.25 μm TLR1, 0.25 μm TLR2, 1.25 μm OspA, 0.05 μm LBP, and/or 0.25 μm sCD14 were incubated for 2 h at 37 °C in a 500-μl volume of PBS buffer, pH 7.4 as indicated. Proteins and protein complexes were separated and analyzed as described in the legend of Fig. 2. The results shown are representative of at least three independent experiments. mAu, milliabsorbance units.

Article Snippet: The unconjugated anti-human TLR1 mAb (clone GD2.F4, CD281) and anti-human TLR2 mAb (clone T2.5, CD282) were obtained from eBioscience (San Diego, CA).

Techniques: Incubation

Journal: The Journal of Biological Chemistry

Article Title: Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex

doi: 10.1074/jbc.M113.453266

Figure Lengend Snippet: Either LBP or soluble CD14 can independently enhance TLR1·TLR2·Pam3CSK4 ternary complex formation. 0.25 μm TLR1, 0.25 μm TLR2, and 2.5 μm Pam3CSK4 were incubated with 0.25 μm BSA (A), 0.05 μm LBP (B), and 0.25 μm sCD14 (C) for 2 h at 37 °C in a 500-μl volume of PBS, pH 7.4 buffer. Proteins and protein complexes were separated and analyzed as described in the legend of Fig. 2. The results shown are representative of at least three independent experiments. mAu, milliabsorbance units.

Article Snippet: The unconjugated anti-human TLR1 mAb (clone GD2.F4, CD281) and anti-human TLR2 mAb (clone T2.5, CD282) were obtained from eBioscience (San Diego, CA).

Techniques: Incubation

Journal: The Journal of Biological Chemistry

Article Title: Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex

doi: 10.1074/jbc.M113.453266

Figure Lengend Snippet: Substoichiometric concentrations of either LBP or soluble CD14 are sufficient to enhance TLR1·TLR2·Pam3CSK4 ternary complex formation. 0.25 μm TLR1, 0.25 μm TLR2, and 2.5 μm Pam3CSK4 were incubated with various concentrations (250, 50, 10, 5, and 0 nm) of either LBP (left panel) or sCD14 (right panel) for 2 h in a 500-μl volume at 37 °C in PBS buffer, pH 7.4. Proteins and protein complexes were separated and analyzed as described in the legend of Fig. 2. The results shown are representative of at least three independent experiments. mAu, milliabsorbance units.

Article Snippet: The unconjugated anti-human TLR1 mAb (clone GD2.F4, CD281) and anti-human TLR2 mAb (clone T2.5, CD282) were obtained from eBioscience (San Diego, CA).

Techniques: Incubation

Journal: The Journal of Biological Chemistry

Article Title: Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex

doi: 10.1074/jbc.M113.453266

Figure Lengend Snippet: Either LBP or soluble CD14 enhances cellular responses to Pam3CSK4 and OspA. HEK 293F cells were co-transfected with vectors expressing full-length TLR1 and TLR2 or empty CMV control vector as indicated together with an NF-κB-promoter driven luciferase reporter gene and a Renilla luciferase reporter gene. About 48 h post-transfection, cells were stimulated with 1 ng/ml Pam3CSK4, OspA, or the non-acylated Ac2CSK4 control in the presence of 0.1 μg/ml LBP, sCD14, or human serum albumin (HSA) as indicated (left side). In one set of experiments, agonists were preincubated with proteins for 1 h at 37 °C prior to addition to transfected cells (right side). Cell values on the y axis represent the level of constitutive reporter activation normalized to the empty CMV vector control (value of 1). Error bars represent the S.D. of three independent values.

Article Snippet: The unconjugated anti-human TLR1 mAb (clone GD2.F4, CD281) and anti-human TLR2 mAb (clone T2.5, CD282) were obtained from eBioscience (San Diego, CA).

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Activation Assay