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  • 94
    Millipore mdp
    Mdp, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mdp/product/Millipore
    Average 94 stars, based on 173 article reviews
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    99
    InvivoGen mdp
    NLRP12 suppresses <t>MDP-induced</t> NF-κB activation by targeting the NOD2/RIPK2 complex for assembling of K48 poly-ubiquitin chains on NOD2 and subsequent degradation. a Western blot analysis of NOD2 ubiquitination in HEK293T cells that were transfected with FLAG-tagged NOD2 in the presence of increasing amounts of NLRP12 as indicated. b Western blot analysis of NOD2 stability in the presence of cycloheximide (CHX at 20 μg/mL) for 4 h. HEK293T cell extracts were subjected to western blot analysis for FLAG-tagged NOD2, Myc-tagged NLRP12 and β-actin. c NF-κB-Luciferase activity in HEK293T cells normalized to expression of β-galactosidase. Depicted are mean ± SEM ( n = 3). d Immunoblot analysis of poly-K48 ubiquitination on NOD2 and of HSP90α/β following co-immunoprecipitation with NOD2 by using the anti-Myc antibody in THP-1 Myc-BirA*-NOD2 cells that were stimulated with MDP (10 µg/mL; left panel) or LPS (0.2 µg/mL, right panel) for 2 h with or without <t>MG132</t> (12.5 µM)
    Mdp, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 506 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mdp  (Bachem)
    92
    Bachem mdp
    The <t>IL-1β</t> autocrine loop contributes to Nod2-mediated induction of pro- and antiinflammatory cytokines in MDMs. A , human MDMs from healthy controls were stimulated with 100 μg/ml <t>MDP</t> or 100 μg/ml MDP with IL-1R signaling blockade (0.5 μg/ml IL-1Ra and 1 μg/ml anti-IL-1β antibody) for 24 h. Supernatants were assayed for TNF-α, IL-8, IL-6, IL-1β, or IL-10. NT , not treated. B , summarized data ( n = 12) are represented as the percent TNF-α, IL-8, IL-6, IL-1β, or IL-10 secretion by cells upon IL-1R signaling blockade normalized to cells in the absence of the blockade (represented by the dotted line at 100%) + S.E. ( error bars ). IL-1β stimulation is included to ensure efficacy of the IL-1R signaling blockade. Significance compared with cells in the absence of the IL-1R blockade is shown. ††, p
    Mdp, supplied by Bachem, used in various techniques. Bioz Stars score: 92/100, based on 167 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    InvivoGen l18 mdp
    RIPK2 kinase‐dead mutants support NOD2 signaling Purification of Ub‐conjugates from THP‐1 cells after treatment with <t>L18‐MDP</t> (200 ng/ml, 1 h) and ponatinib or GSK583 as indicated. Purified material and lysates were analyzed by immunoblotting. Schematic representation of RIPK2 mRNA and protein with indicated positions of RIPK2 gRNA sequences (CRISPR/Cas9 B and C) and epitopes for RIPK2 antibodies used in this study. E1–E11 indicates RIPK2 exons. Identification RIPK2 knockout U2OS/NOD2 cell clones grown from cell cultures transfected with CRISPR/Cas9 vectors targeting RIPK2. Clones B7‐1 and C5‐2 were used in the study. Radioactive in vitro RIPK2 kinase assay with RIPK2 variants expressed in U2OS/NOD2 RIPK2 KO cells and purified with anti‐HA. The in vitro phosphorylated RIPK2 and universal kinase substrate MBP were separated by SDS–PAGE and exposed to X‐ray film. The inputs and precipitated proteins were analyzed by immunoblotting. Intracellular flow cytometry analysis of CXCL8 following L18‐MDP treatment (200 ng/ml, 4 h) of U2OS/NOD2 RIPK2 KO cells (clone B7‐1) reconstituted with RIPK2 variants or empty vector as indicated. Data information: Data represent the mean ± SEM of at least three independent experiments. * P
    L18 Mdp, supplied by InvivoGen, used in various techniques. Bioz Stars score: 95/100, based on 133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Pharmaxis mdp challenge
    % difference of <t>FENO</t> after <t>MDP</t> challenge by baseline FENO, asthma, atopy and BHR severity. Abbreviations: FENO, fractional exhaled nitric oxide; MDP, mannitol dry powder; BHR, bronchial hyperresponsiveness; SPT, skin prick test. Baseline FENO defined as tertiles (low levels:
    Mdp Challenge, supplied by Pharmaxis, used in various techniques. Bioz Stars score: 88/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher mdp
    % difference of <t>FENO</t> after <t>MDP</t> challenge by baseline FENO, asthma, atopy and BHR severity. Abbreviations: FENO, fractional exhaled nitric oxide; MDP, mannitol dry powder; BHR, bronchial hyperresponsiveness; SPT, skin prick test. Baseline FENO defined as tertiles (low levels:
    Mdp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Bachem synthetic mdp
    Enhanced Nod1-induced cytokine production and signaling in Nod2-deficient macrophages pre-exposed to <t>LPS</t> and <t>MDP.</t> A and B , BMDMs from wild-type and Nod2 -/- mice were treated with combination of LPS and MDP or left untreated for 24 hrs. The macrophages
    Synthetic Mdp, supplied by Bachem, used in various techniques. Bioz Stars score: 88/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    3M Co mdp free primer
    Enhanced Nod1-induced cytokine production and signaling in Nod2-deficient macrophages pre-exposed to <t>LPS</t> and <t>MDP.</t> A and B , BMDMs from wild-type and Nod2 -/- mice were treated with combination of LPS and MDP or left untreated for 24 hrs. The macrophages
    Mdp Free Primer, supplied by 3M Co, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Bachem muramyl dipeptide mdp
    Enhanced Nod1-induced cytokine production and signaling in Nod2-deficient macrophages pre-exposed to <t>LPS</t> and <t>MDP.</t> A and B , BMDMs from wild-type and Nod2 -/- mice were treated with combination of LPS and MDP or left untreated for 24 hrs. The macrophages
    Muramyl Dipeptide Mdp, supplied by Bachem, used in various techniques. Bioz Stars score: 90/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    InvivoGen mdp rhodamine
    GSK669 exhibits NOD2 selectivity in cells that express endogenous NOD1 and NOD2. Concentration response curves of GSK669 and its close analogue GSK400 for the inhibition of <t>MDP</t> (A) but not Tri-DAP (B) stimulated IL-8 secretion in HCT116 cells which express functionally active NOD1 and NOD2. Cells were pre-incubated with compounds for 1 hour prior to addition of <t>NOD</t> agonists. IL-8 secreted into medium was assayed after 24 hours. The same RIP2 inhibitor used in Figure 2C was included as a positive control. Data are the average percent inhibition obtained from 1 (RIP2), 2 (GSK400) or 4 (GSK669) separate experiments.
    Mdp Rhodamine, supplied by InvivoGen, used in various techniques. Bioz Stars score: 85/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Siemens Healthineers 99m tc mdp
    ROC curve analysis between the histologic response and each parameter of the 18 F-FDG PET and <t>99m</t> <t>Tc-MDP</t> bone scans. In ROC curves using the 18 F-FDG PET parameters (A), Δ%SUV max shows the largest AUC value (0.829). The AUC values for SUV max 1 and SUV max 2 are 0.571 and 0.817, respectively. In ROC curves using 99m Tc-MDP bone scan parameters (B), the AUC for Δ%T/NT max has the largest value (0.772). The AUC values for T/NT max 1 and T/NT max 2 are 0.601 and 0.770, respectively. The ROC curves regarding Δ%SUV max and Δ%T/NT max are compared (C). Two curves did not show significant difference ( P = 0.44). 18 F-FDG PET = 18 F-Fluorodeoxyglucose positron emission tomography, 99m Tc-MDP = 99m Tc-methyl diphosphonate, Δ%SUV max = percent changes of the maximum standardized uptake value, Δ%T/NT max = percent changes of the maximum tumor-to-nontumor ratio, AUC = area under the curve, ROC = receiver operating characteristic.
    99m Tc Mdp, supplied by Siemens Healthineers, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Instrum GmbH microwave detected photoconductivity mdp map
    ROC curve analysis between the histologic response and each parameter of the 18 F-FDG PET and <t>99m</t> <t>Tc-MDP</t> bone scans. In ROC curves using the 18 F-FDG PET parameters (A), Δ%SUV max shows the largest AUC value (0.829). The AUC values for SUV max 1 and SUV max 2 are 0.571 and 0.817, respectively. In ROC curves using 99m Tc-MDP bone scan parameters (B), the AUC for Δ%T/NT max has the largest value (0.772). The AUC values for T/NT max 1 and T/NT max 2 are 0.601 and 0.770, respectively. The ROC curves regarding Δ%SUV max and Δ%T/NT max are compared (C). Two curves did not show significant difference ( P = 0.44). 18 F-FDG PET = 18 F-Fluorodeoxyglucose positron emission tomography, 99m Tc-MDP = 99m Tc-methyl diphosphonate, Δ%SUV max = percent changes of the maximum standardized uptake value, Δ%T/NT max = percent changes of the maximum tumor-to-nontumor ratio, AUC = area under the curve, ROC = receiver operating characteristic.
    Microwave Detected Photoconductivity Mdp Map, supplied by Instrum GmbH, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/microwave detected photoconductivity mdp map/product/Instrum GmbH
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    92
    InvivoGen mdp control
    <t>iE-DAP,</t> <t>MDP</t> and Poly(I:C)LyoVec induce T-cell proliferation
    Mdp Control, supplied by InvivoGen, used in various techniques. Bioz Stars score: 92/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    3M Co mdp free resin cement
    <t>iE-DAP,</t> <t>MDP</t> and Poly(I:C)LyoVec induce T-cell proliferation
    Mdp Free Resin Cement, supplied by 3M Co, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    InvivoGen n glycolyl mdp
    <t>iE-DAP,</t> <t>MDP</t> and Poly(I:C)LyoVec induce T-cell proliferation
    N Glycolyl Mdp, supplied by InvivoGen, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    InvivoGen mdp dd
    NLRP12 suppresses <t>MDP-induced</t> NF-κB activation by targeting the NOD2/RIPK2 complex for assembling of K48 poly-ubiquitin chains on NOD2 and subsequent degradation. a Western blot analysis of NOD2 ubiquitination in HEK293T cells that were transfected with FLAG-tagged NOD2 in the presence of increasing amounts of NLRP12 as indicated. b Western blot analysis of NOD2 stability in the presence of cycloheximide (CHX at 20 μg/mL) for 4 h. HEK293T cell extracts were subjected to western blot analysis for FLAG-tagged NOD2, Myc-tagged NLRP12 and β-actin. c NF-κB-Luciferase activity in HEK293T cells normalized to expression of β-galactosidase. Depicted are mean ± SEM ( n = 3). d Immunoblot analysis of poly-K48 ubiquitination on NOD2 and of HSP90α/β following co-immunoprecipitation with NOD2 by using the anti-Myc antibody in THP-1 Myc-BirA*-NOD2 cells that were stimulated with MDP (10 µg/mL; left panel) or <t>LPS</t> (0.2 µg/mL, right panel) for 2 h with or without MG132 (12.5 µM)
    Mdp Dd, supplied by InvivoGen, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Kuraray Medical Inc 10 mdp
    NLRP12 suppresses <t>MDP-induced</t> NF-κB activation by targeting the NOD2/RIPK2 complex for assembling of K48 poly-ubiquitin chains on NOD2 and subsequent degradation. a Western blot analysis of NOD2 ubiquitination in HEK293T cells that were transfected with FLAG-tagged NOD2 in the presence of increasing amounts of NLRP12 as indicated. b Western blot analysis of NOD2 stability in the presence of cycloheximide (CHX at 20 μg/mL) for 4 h. HEK293T cell extracts were subjected to western blot analysis for FLAG-tagged NOD2, Myc-tagged NLRP12 and β-actin. c NF-κB-Luciferase activity in HEK293T cells normalized to expression of β-galactosidase. Depicted are mean ± SEM ( n = 3). d Immunoblot analysis of poly-K48 ubiquitination on NOD2 and of HSP90α/β following co-immunoprecipitation with NOD2 by using the anti-Myc antibody in THP-1 Myc-BirA*-NOD2 cells that were stimulated with MDP (10 µg/mL; left panel) or <t>LPS</t> (0.2 µg/mL, right panel) for 2 h with or without MG132 (12.5 µM)
    10 Mdp, supplied by Kuraray Medical Inc, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    NLRP12 suppresses MDP-induced NF-κB activation by targeting the NOD2/RIPK2 complex for assembling of K48 poly-ubiquitin chains on NOD2 and subsequent degradation. a Western blot analysis of NOD2 ubiquitination in HEK293T cells that were transfected with FLAG-tagged NOD2 in the presence of increasing amounts of NLRP12 as indicated. b Western blot analysis of NOD2 stability in the presence of cycloheximide (CHX at 20 μg/mL) for 4 h. HEK293T cell extracts were subjected to western blot analysis for FLAG-tagged NOD2, Myc-tagged NLRP12 and β-actin. c NF-κB-Luciferase activity in HEK293T cells normalized to expression of β-galactosidase. Depicted are mean ± SEM ( n = 3). d Immunoblot analysis of poly-K48 ubiquitination on NOD2 and of HSP90α/β following co-immunoprecipitation with NOD2 by using the anti-Myc antibody in THP-1 Myc-BirA*-NOD2 cells that were stimulated with MDP (10 µg/mL; left panel) or LPS (0.2 µg/mL, right panel) for 2 h with or without MG132 (12.5 µM)

    Journal: Nature Communications

    Article Title: Proteasomal degradation of NOD2 by NLRP12 in monocytes promotes bacterial tolerance and colonization by enteropathogens

    doi: 10.1038/s41467-018-07750-5

    Figure Lengend Snippet: NLRP12 suppresses MDP-induced NF-κB activation by targeting the NOD2/RIPK2 complex for assembling of K48 poly-ubiquitin chains on NOD2 and subsequent degradation. a Western blot analysis of NOD2 ubiquitination in HEK293T cells that were transfected with FLAG-tagged NOD2 in the presence of increasing amounts of NLRP12 as indicated. b Western blot analysis of NOD2 stability in the presence of cycloheximide (CHX at 20 μg/mL) for 4 h. HEK293T cell extracts were subjected to western blot analysis for FLAG-tagged NOD2, Myc-tagged NLRP12 and β-actin. c NF-κB-Luciferase activity in HEK293T cells normalized to expression of β-galactosidase. Depicted are mean ± SEM ( n = 3). d Immunoblot analysis of poly-K48 ubiquitination on NOD2 and of HSP90α/β following co-immunoprecipitation with NOD2 by using the anti-Myc antibody in THP-1 Myc-BirA*-NOD2 cells that were stimulated with MDP (10 µg/mL; left panel) or LPS (0.2 µg/mL, right panel) for 2 h with or without MG132 (12.5 µM)

    Article Snippet: THP-1 Myc-BirA*-NOD2 cells were pre-treated for one h with DMSO (vehicle) or MG132 (at 12.5 µM) before 10 µg/mL of MDP (InvivoGen, France) was added.

    Techniques: Activation Assay, Western Blot, Transfection, Luciferase, Activity Assay, Expressing, Immunoprecipitation

    SipA-mediated NF-κB activation requires both NOD1 and NOD2 in vitro . (A to C) HEK293 cells were transfected with the NF-κB luciferase reporter construct and hNOD1DN, hNOD2DN, hRIP2DN, or vector only and stimulated with 1 µg/ml C12-iE-DAP (NOD1 ligand) (A), 10 µg/ml MDP (NOD2 ligand) (B), and 100 ng/ml purified flagellin (C). (D and E) HEK293 cells were cotransfected with the NF-κB luciferase reporter construct and GFP-SipA (D) or GFP-SipA425 (E) and hNOD1DN, hNOD2DN, hRIP2DN, or vector only. Data are expressed as percent NF-κB inhibition, where 100% was set to be the response in the cells transfected with vector only. Values represent the means ± standard errors of at least three independent experiments. Brackets indicate the statistical differences. NS, not significant. (F) Western blot analysis of HEK293 cells expressing GFP-tagged SipA425 and Flag-tagged hNOD1DN, Flag-tagged hNOD2DN, HA-tagged hRIP2DN, or vector only. Cotransfection did not influence the expression of GFP-SipA425.

    Journal: mBio

    Article Title: A Salmonella Virulence Factor Activates the NOD1/NOD2 Signaling Pathway

    doi: 10.1128/mBio.00266-11

    Figure Lengend Snippet: SipA-mediated NF-κB activation requires both NOD1 and NOD2 in vitro . (A to C) HEK293 cells were transfected with the NF-κB luciferase reporter construct and hNOD1DN, hNOD2DN, hRIP2DN, or vector only and stimulated with 1 µg/ml C12-iE-DAP (NOD1 ligand) (A), 10 µg/ml MDP (NOD2 ligand) (B), and 100 ng/ml purified flagellin (C). (D and E) HEK293 cells were cotransfected with the NF-κB luciferase reporter construct and GFP-SipA (D) or GFP-SipA425 (E) and hNOD1DN, hNOD2DN, hRIP2DN, or vector only. Data are expressed as percent NF-κB inhibition, where 100% was set to be the response in the cells transfected with vector only. Values represent the means ± standard errors of at least three independent experiments. Brackets indicate the statistical differences. NS, not significant. (F) Western blot analysis of HEK293 cells expressing GFP-tagged SipA425 and Flag-tagged hNOD1DN, Flag-tagged hNOD2DN, HA-tagged hRIP2DN, or vector only. Cotransfection did not influence the expression of GFP-SipA425.

    Article Snippet: After 48 h of incubation, the cells were infected with the appropriate bacterial strains or the NOD1 ligand C12-iE-DAP, the NOD2 ligand MDP, or the TLR5 ligand flagellin (InvivoGen).

    Techniques: Activation Assay, In Vitro, Transfection, Luciferase, Construct, Plasmid Preparation, Purification, Inhibition, Western Blot, Expressing, Cotransfection

    TLR and NOD signaling are both required for an efficient macrophage-mediated intracellular bacterial killing. Peritoneal macrophages isolated from wild-type, TLR4- and TLR2-deficient, and NOD1- and NOD2-deficient mice were stimulated with LPS (10 ng/ml), Tri-DAP (5 μg/ml), MDP (5 μg/ml), and their combinations (A) or BLP (10 ng/ml), Tri-DAP (5 μg/ml), MDP (5 μg/ml), and their combinations (B) for 6 h, and further incubated with live S. typhimurium ( S. typhi ) (A) or live S. aureus (B) for 60 min to assess intracellular bacterial killing. Data are expressed as mean ± SD from four to five independent experiments in triplicate. * p

    Journal: Frontiers in Immunology

    Article Title: Activation of Both TLR and NOD Signaling Confers Host Innate Immunity-Mediated Protection Against Microbial Infection

    doi: 10.3389/fimmu.2018.03082

    Figure Lengend Snippet: TLR and NOD signaling are both required for an efficient macrophage-mediated intracellular bacterial killing. Peritoneal macrophages isolated from wild-type, TLR4- and TLR2-deficient, and NOD1- and NOD2-deficient mice were stimulated with LPS (10 ng/ml), Tri-DAP (5 μg/ml), MDP (5 μg/ml), and their combinations (A) or BLP (10 ng/ml), Tri-DAP (5 μg/ml), MDP (5 μg/ml), and their combinations (B) for 6 h, and further incubated with live S. typhimurium ( S. typhi ) (A) or live S. aureus (B) for 60 min to assess intracellular bacterial killing. Data are expressed as mean ± SD from four to five independent experiments in triplicate. * p

    Article Snippet: The NOD1 agonist L-Ala-γ-D-Glu-mDAP (Tri-DAP) and NOD2 agonist MDP were obtained from InvivoGen.

    Techniques: Isolation, Mouse Assay, Incubation

    The augmented inflammatory response is dependent on the intact of both TLR and NOD signaling. Peritoneal macrophages and BMMs isolated from wild-type, TLR4- and TLR2-deficient, and NOD1- and NOD2-deficient mice were stimulated with LPS (10 ng/ml), Tri-DAP (5 μg/ml), MDP (5 μg/ml), and their combinations (A) or BLP (10 ng/ml), Tri-DAP (5 μg/ml), MDP (5 μg/ml), and their combinations (B) for 12 h. Macrophages incubated with PBS were used as the control. TNF-α concentrations in the supernatants were assessed by cytometric bead array. Data are expressed as mean ± SD from five to six independent experiments in duplicate. * p

    Journal: Frontiers in Immunology

    Article Title: Activation of Both TLR and NOD Signaling Confers Host Innate Immunity-Mediated Protection Against Microbial Infection

    doi: 10.3389/fimmu.2018.03082

    Figure Lengend Snippet: The augmented inflammatory response is dependent on the intact of both TLR and NOD signaling. Peritoneal macrophages and BMMs isolated from wild-type, TLR4- and TLR2-deficient, and NOD1- and NOD2-deficient mice were stimulated with LPS (10 ng/ml), Tri-DAP (5 μg/ml), MDP (5 μg/ml), and their combinations (A) or BLP (10 ng/ml), Tri-DAP (5 μg/ml), MDP (5 μg/ml), and their combinations (B) for 12 h. Macrophages incubated with PBS were used as the control. TNF-α concentrations in the supernatants were assessed by cytometric bead array. Data are expressed as mean ± SD from five to six independent experiments in duplicate. * p

    Article Snippet: The NOD1 agonist L-Ala-γ-D-Glu-mDAP (Tri-DAP) and NOD2 agonist MDP were obtained from InvivoGen.

    Techniques: Isolation, Mouse Assay, Incubation

    Stimulation of macrophages with the combined TLR and NOD agonists amplifies downstream NF-κB activation and augments NF-κB p65 binding to TNF-α and IL-6 promoters. Isolated BMMs were stimulated with LPS (10 ng/ml), Tri-DAP (5 μg/ml), MDP (5 μg/ml), and their combinations either for 30 min (A) or for the indicated time periods (B,C) . Cytoplasmic proteins were extracted and subjected to immunoblotting for detection of either TLR4, NOD1, NOD2, MyD88, IRAK1, RIP2, and CARD9 (A) or total and phosphorylated p65 (P-p65), total and phosphorylated IκBα (P- IκBα), and total and phosphorylated p38 (P-p38) (B,C) . Results shown represent one experiment from a total of three to four separate experiments. The intensity of P-65, total IκBα, P-IκBα, and P-p38 signal in each band was normalized by GAPDH (D,E) . Data are expressed as mean ± SD from three to four separate experiments. * p

    Journal: Frontiers in Immunology

    Article Title: Activation of Both TLR and NOD Signaling Confers Host Innate Immunity-Mediated Protection Against Microbial Infection

    doi: 10.3389/fimmu.2018.03082

    Figure Lengend Snippet: Stimulation of macrophages with the combined TLR and NOD agonists amplifies downstream NF-κB activation and augments NF-κB p65 binding to TNF-α and IL-6 promoters. Isolated BMMs were stimulated with LPS (10 ng/ml), Tri-DAP (5 μg/ml), MDP (5 μg/ml), and their combinations either for 30 min (A) or for the indicated time periods (B,C) . Cytoplasmic proteins were extracted and subjected to immunoblotting for detection of either TLR4, NOD1, NOD2, MyD88, IRAK1, RIP2, and CARD9 (A) or total and phosphorylated p65 (P-p65), total and phosphorylated IκBα (P- IκBα), and total and phosphorylated p38 (P-p38) (B,C) . Results shown represent one experiment from a total of three to four separate experiments. The intensity of P-65, total IκBα, P-IκBα, and P-p38 signal in each band was normalized by GAPDH (D,E) . Data are expressed as mean ± SD from three to four separate experiments. * p

    Article Snippet: The NOD1 agonist L-Ala-γ-D-Glu-mDAP (Tri-DAP) and NOD2 agonist MDP were obtained from InvivoGen.

    Techniques: Activation Assay, Binding Assay, Isolation

    The IL-1β autocrine loop contributes to Nod2-mediated induction of pro- and antiinflammatory cytokines in MDMs. A , human MDMs from healthy controls were stimulated with 100 μg/ml MDP or 100 μg/ml MDP with IL-1R signaling blockade (0.5 μg/ml IL-1Ra and 1 μg/ml anti-IL-1β antibody) for 24 h. Supernatants were assayed for TNF-α, IL-8, IL-6, IL-1β, or IL-10. NT , not treated. B , summarized data ( n = 12) are represented as the percent TNF-α, IL-8, IL-6, IL-1β, or IL-10 secretion by cells upon IL-1R signaling blockade normalized to cells in the absence of the blockade (represented by the dotted line at 100%) + S.E. ( error bars ). IL-1β stimulation is included to ensure efficacy of the IL-1R signaling blockade. Significance compared with cells in the absence of the IL-1R blockade is shown. ††, p

    Journal: The Journal of Biological Chemistry

    Article Title: Distinct Roles for Nod2 Protein and Autocrine Interleukin-1? in Muramyl Dipeptide-induced Mitogen-activated Protein Kinase Activation and Cytokine Secretion in Human Macrophages *

    doi: 10.1074/jbc.M111.237495

    Figure Lengend Snippet: The IL-1β autocrine loop contributes to Nod2-mediated induction of pro- and antiinflammatory cytokines in MDMs. A , human MDMs from healthy controls were stimulated with 100 μg/ml MDP or 100 μg/ml MDP with IL-1R signaling blockade (0.5 μg/ml IL-1Ra and 1 μg/ml anti-IL-1β antibody) for 24 h. Supernatants were assayed for TNF-α, IL-8, IL-6, IL-1β, or IL-10. NT , not treated. B , summarized data ( n = 12) are represented as the percent TNF-α, IL-8, IL-6, IL-1β, or IL-10 secretion by cells upon IL-1R signaling blockade normalized to cells in the absence of the blockade (represented by the dotted line at 100%) + S.E. ( error bars ). IL-1β stimulation is included to ensure efficacy of the IL-1R signaling blockade. Significance compared with cells in the absence of the IL-1R blockade is shown. ††, p

    Article Snippet: For IL-1β blockade, IL-1R antagonist (IL-1Ra) (GenScript, Piscataway, NJ) and/or anti-IL-1β-blocking antibody (R & D Systems, Minneapolis, MN) was added 1 h prior to treatments with MDP (Bachem, King of Prussia, PA), IL-1β (eBioscience, San Diego, CA), lipid A (Peptides International, Louisville, KY), poly(I:C), CpG DNA (Calbiochem), TriDAP, flagellin, or CL097 (Invivogen).

    Techniques:

    Decreased MAPK activation upon Nod2 signaling in the absence of the IL-1β autocrine loop persists over time. Human MDMs from healthy controls ( n = 6–8) were stimulated with 100 μg/ml MDP, 10 ng/ml IL-1β, or 100 μg/ml MDP with 0.5 μg/ml IL-1Ra and 1 μg/ml anti-IL-1β antibody for 10, 30, or 60 min and analyzed by flow cytometry for the expression of phospho-JNK, phospho-ERK, or phospho-p38. A , shown are representative flow cytometry plots with the indicated mean fluorescence intensity values. Stimulated cells stained with isotype controls are shown. B , summarized data are represented as the -fold phospho-MAPK induction normalized to untreated cells + S.E. ( error bars ). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Distinct Roles for Nod2 Protein and Autocrine Interleukin-1? in Muramyl Dipeptide-induced Mitogen-activated Protein Kinase Activation and Cytokine Secretion in Human Macrophages *

    doi: 10.1074/jbc.M111.237495

    Figure Lengend Snippet: Decreased MAPK activation upon Nod2 signaling in the absence of the IL-1β autocrine loop persists over time. Human MDMs from healthy controls ( n = 6–8) were stimulated with 100 μg/ml MDP, 10 ng/ml IL-1β, or 100 μg/ml MDP with 0.5 μg/ml IL-1Ra and 1 μg/ml anti-IL-1β antibody for 10, 30, or 60 min and analyzed by flow cytometry for the expression of phospho-JNK, phospho-ERK, or phospho-p38. A , shown are representative flow cytometry plots with the indicated mean fluorescence intensity values. Stimulated cells stained with isotype controls are shown. B , summarized data are represented as the -fold phospho-MAPK induction normalized to untreated cells + S.E. ( error bars ). *, p

    Article Snippet: For IL-1β blockade, IL-1R antagonist (IL-1Ra) (GenScript, Piscataway, NJ) and/or anti-IL-1β-blocking antibody (R & D Systems, Minneapolis, MN) was added 1 h prior to treatments with MDP (Bachem, King of Prussia, PA), IL-1β (eBioscience, San Diego, CA), lipid A (Peptides International, Louisville, KY), poly(I:C), CpG DNA (Calbiochem), TriDAP, flagellin, or CL097 (Invivogen).

    Techniques: Activation Assay, Flow Cytometry, Cytometry, Expressing, Fluorescence, Staining

    The IL-1β autocrine loop regulates cytokine mRNA upon MDP stimulation of MDMs. Human MDMs from healthy controls ( n = 12) were stimulated with 100 μg/ml MDP, 10 ng/ml IL-1β, or 100 μg/ml MDP with 0.5 μg/ml IL-1Ra and 1 μg/ml anti-IL-1β antibody for 4 h. TNF-α, IL-8, and IL-1β mRNA expression were assessed by real-time PCR. Data are represented as the -fold induction of cytokine mRNA compared with untreated cells (represented by the dotted line at 1) + S.E. ( error bars ). **, p

    Journal: The Journal of Biological Chemistry

    Article Title: Distinct Roles for Nod2 Protein and Autocrine Interleukin-1? in Muramyl Dipeptide-induced Mitogen-activated Protein Kinase Activation and Cytokine Secretion in Human Macrophages *

    doi: 10.1074/jbc.M111.237495

    Figure Lengend Snippet: The IL-1β autocrine loop regulates cytokine mRNA upon MDP stimulation of MDMs. Human MDMs from healthy controls ( n = 12) were stimulated with 100 μg/ml MDP, 10 ng/ml IL-1β, or 100 μg/ml MDP with 0.5 μg/ml IL-1Ra and 1 μg/ml anti-IL-1β antibody for 4 h. TNF-α, IL-8, and IL-1β mRNA expression were assessed by real-time PCR. Data are represented as the -fold induction of cytokine mRNA compared with untreated cells (represented by the dotted line at 1) + S.E. ( error bars ). **, p

    Article Snippet: For IL-1β blockade, IL-1R antagonist (IL-1Ra) (GenScript, Piscataway, NJ) and/or anti-IL-1β-blocking antibody (R & D Systems, Minneapolis, MN) was added 1 h prior to treatments with MDP (Bachem, King of Prussia, PA), IL-1β (eBioscience, San Diego, CA), lipid A (Peptides International, Louisville, KY), poly(I:C), CpG DNA (Calbiochem), TriDAP, flagellin, or CL097 (Invivogen).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    IL-1β stimulation of MDMs demonstrates earlier MAPK activation compared with MDP stimulation. Human MDMs from healthy controls ( n = 6–8) were stimulated with 100 μg/ml MDP or 10 ng/ml IL-1β for 5 and 10 min and analyzed by flow cytometry for the expression of phospho-JNK, phospho-ERK, or phospho-p38. A , shown are representative flow cytometry plots with indicated mean fluorescence intensity values. Stimulated cells stained with isotype controls are shown. B , summarized data are represented as the -fold phospho-MAPK induction normalized to untreated cells + S.E. ( error bars ). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Distinct Roles for Nod2 Protein and Autocrine Interleukin-1? in Muramyl Dipeptide-induced Mitogen-activated Protein Kinase Activation and Cytokine Secretion in Human Macrophages *

    doi: 10.1074/jbc.M111.237495

    Figure Lengend Snippet: IL-1β stimulation of MDMs demonstrates earlier MAPK activation compared with MDP stimulation. Human MDMs from healthy controls ( n = 6–8) were stimulated with 100 μg/ml MDP or 10 ng/ml IL-1β for 5 and 10 min and analyzed by flow cytometry for the expression of phospho-JNK, phospho-ERK, or phospho-p38. A , shown are representative flow cytometry plots with indicated mean fluorescence intensity values. Stimulated cells stained with isotype controls are shown. B , summarized data are represented as the -fold phospho-MAPK induction normalized to untreated cells + S.E. ( error bars ). *, p

    Article Snippet: For IL-1β blockade, IL-1R antagonist (IL-1Ra) (GenScript, Piscataway, NJ) and/or anti-IL-1β-blocking antibody (R & D Systems, Minneapolis, MN) was added 1 h prior to treatments with MDP (Bachem, King of Prussia, PA), IL-1β (eBioscience, San Diego, CA), lipid A (Peptides International, Louisville, KY), poly(I:C), CpG DNA (Calbiochem), TriDAP, flagellin, or CL097 (Invivogen).

    Techniques: Activation Assay, Flow Cytometry, Cytometry, Expressing, Fluorescence, Staining

    Model for the role of autocrine IL-1β in MAPK and cytokine induction upon Nod2 stimulation in primary MDMs. MDP stimulation of Nod2 activates caspase-1 to process pro-IL-1β stores that rapidly feed back in an autocrine fashion to stimulate the IL-1R and substantially increase MAPK phosphorylation. This increased MAPK activation significantly enhances secretion of both anti- and proinflammatory cytokines.

    Journal: The Journal of Biological Chemistry

    Article Title: Distinct Roles for Nod2 Protein and Autocrine Interleukin-1? in Muramyl Dipeptide-induced Mitogen-activated Protein Kinase Activation and Cytokine Secretion in Human Macrophages *

    doi: 10.1074/jbc.M111.237495

    Figure Lengend Snippet: Model for the role of autocrine IL-1β in MAPK and cytokine induction upon Nod2 stimulation in primary MDMs. MDP stimulation of Nod2 activates caspase-1 to process pro-IL-1β stores that rapidly feed back in an autocrine fashion to stimulate the IL-1R and substantially increase MAPK phosphorylation. This increased MAPK activation significantly enhances secretion of both anti- and proinflammatory cytokines.

    Article Snippet: For IL-1β blockade, IL-1R antagonist (IL-1Ra) (GenScript, Piscataway, NJ) and/or anti-IL-1β-blocking antibody (R & D Systems, Minneapolis, MN) was added 1 h prior to treatments with MDP (Bachem, King of Prussia, PA), IL-1β (eBioscience, San Diego, CA), lipid A (Peptides International, Louisville, KY), poly(I:C), CpG DNA (Calbiochem), TriDAP, flagellin, or CL097 (Invivogen).

    Techniques: Activation Assay

    Selective activation of JNK and p38 rescues the decreased cytokine secretion observed during Nod2 signaling in the absence of the IL-1β autocrine loop. A and B , human MDMs from healthy controls ( n = 12) were stimulated with 100 μg/ml MDP or 100 μg/ml MDP with 0.5 μg/ml IL-1Ra and 1 μg/ml anti-IL-1β antibody in the presence or absence of 50 ng/ml anisomycin. A , shown are representative flow cytometry plots with indicated mean fluorescence intensity values. B , data are represented as the -fold MAPK activation normalized to untreated cells + S.E. ( error bars ). **, p

    Journal: The Journal of Biological Chemistry

    Article Title: Distinct Roles for Nod2 Protein and Autocrine Interleukin-1? in Muramyl Dipeptide-induced Mitogen-activated Protein Kinase Activation and Cytokine Secretion in Human Macrophages *

    doi: 10.1074/jbc.M111.237495

    Figure Lengend Snippet: Selective activation of JNK and p38 rescues the decreased cytokine secretion observed during Nod2 signaling in the absence of the IL-1β autocrine loop. A and B , human MDMs from healthy controls ( n = 12) were stimulated with 100 μg/ml MDP or 100 μg/ml MDP with 0.5 μg/ml IL-1Ra and 1 μg/ml anti-IL-1β antibody in the presence or absence of 50 ng/ml anisomycin. A , shown are representative flow cytometry plots with indicated mean fluorescence intensity values. B , data are represented as the -fold MAPK activation normalized to untreated cells + S.E. ( error bars ). **, p

    Article Snippet: For IL-1β blockade, IL-1R antagonist (IL-1Ra) (GenScript, Piscataway, NJ) and/or anti-IL-1β-blocking antibody (R & D Systems, Minneapolis, MN) was added 1 h prior to treatments with MDP (Bachem, King of Prussia, PA), IL-1β (eBioscience, San Diego, CA), lipid A (Peptides International, Louisville, KY), poly(I:C), CpG DNA (Calbiochem), TriDAP, flagellin, or CL097 (Invivogen).

    Techniques: Activation Assay, Flow Cytometry, Cytometry, Fluorescence

    MDP treatment of MDMs leads to early IL-1β secretion. Human MDMs from healthy controls were stimulated with 100 μg/ml MDP ( n = 8) ( A ) or 100 μg/ml MDP with IL-1R signaling blockade (0.5 μg/ml IL-1Ra and 1 μg/ml anti-IL-1β antibody) ( n = 8) ( B ) for the indicated times. Data are represented as the cytokine concentrations in supernatants at the indicated time points + S.E. ( error bars ). Significance compared with no treatment is shown. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Distinct Roles for Nod2 Protein and Autocrine Interleukin-1? in Muramyl Dipeptide-induced Mitogen-activated Protein Kinase Activation and Cytokine Secretion in Human Macrophages *

    doi: 10.1074/jbc.M111.237495

    Figure Lengend Snippet: MDP treatment of MDMs leads to early IL-1β secretion. Human MDMs from healthy controls were stimulated with 100 μg/ml MDP ( n = 8) ( A ) or 100 μg/ml MDP with IL-1R signaling blockade (0.5 μg/ml IL-1Ra and 1 μg/ml anti-IL-1β antibody) ( n = 8) ( B ) for the indicated times. Data are represented as the cytokine concentrations in supernatants at the indicated time points + S.E. ( error bars ). Significance compared with no treatment is shown. *, p

    Article Snippet: For IL-1β blockade, IL-1R antagonist (IL-1Ra) (GenScript, Piscataway, NJ) and/or anti-IL-1β-blocking antibody (R & D Systems, Minneapolis, MN) was added 1 h prior to treatments with MDP (Bachem, King of Prussia, PA), IL-1β (eBioscience, San Diego, CA), lipid A (Peptides International, Louisville, KY), poly(I:C), CpG DNA (Calbiochem), TriDAP, flagellin, or CL097 (Invivogen).

    Techniques:

    The IL-1β autocrine loop contributes to the majority of MAPK activation upon MDP treatment of MDMs. Human MDMs from healthy controls ( n = 19–23) were stimulated with 100 μg/ml MDP, 10 ng/ml IL-1β, or 100 μg/ml MDP with 0.5 μg/ml IL-1Ra and 1 μg/ml anti-IL-1β antibody for 10 min and analyzed by flow cytometry for the expression of phospho-JNK, phospho-ERK, or phospho-p38. A , shown are representative flow cytometry plots with mean fluorescence intensity values indicated. Stimulated cells stained with isotype controls are shown. B , summarized data are represented as the -fold phospho-MAPK induction normalized to untreated cells + S.E. ( error bars ). ††, p

    Journal: The Journal of Biological Chemistry

    Article Title: Distinct Roles for Nod2 Protein and Autocrine Interleukin-1? in Muramyl Dipeptide-induced Mitogen-activated Protein Kinase Activation and Cytokine Secretion in Human Macrophages *

    doi: 10.1074/jbc.M111.237495

    Figure Lengend Snippet: The IL-1β autocrine loop contributes to the majority of MAPK activation upon MDP treatment of MDMs. Human MDMs from healthy controls ( n = 19–23) were stimulated with 100 μg/ml MDP, 10 ng/ml IL-1β, or 100 μg/ml MDP with 0.5 μg/ml IL-1Ra and 1 μg/ml anti-IL-1β antibody for 10 min and analyzed by flow cytometry for the expression of phospho-JNK, phospho-ERK, or phospho-p38. A , shown are representative flow cytometry plots with mean fluorescence intensity values indicated. Stimulated cells stained with isotype controls are shown. B , summarized data are represented as the -fold phospho-MAPK induction normalized to untreated cells + S.E. ( error bars ). ††, p

    Article Snippet: For IL-1β blockade, IL-1R antagonist (IL-1Ra) (GenScript, Piscataway, NJ) and/or anti-IL-1β-blocking antibody (R & D Systems, Minneapolis, MN) was added 1 h prior to treatments with MDP (Bachem, King of Prussia, PA), IL-1β (eBioscience, San Diego, CA), lipid A (Peptides International, Louisville, KY), poly(I:C), CpG DNA (Calbiochem), TriDAP, flagellin, or CL097 (Invivogen).

    Techniques: Activation Assay, Flow Cytometry, Cytometry, Expressing, Fluorescence, Staining

    RIPK2 kinase‐dead mutants support NOD2 signaling Purification of Ub‐conjugates from THP‐1 cells after treatment with L18‐MDP (200 ng/ml, 1 h) and ponatinib or GSK583 as indicated. Purified material and lysates were analyzed by immunoblotting. Schematic representation of RIPK2 mRNA and protein with indicated positions of RIPK2 gRNA sequences (CRISPR/Cas9 B and C) and epitopes for RIPK2 antibodies used in this study. E1–E11 indicates RIPK2 exons. Identification RIPK2 knockout U2OS/NOD2 cell clones grown from cell cultures transfected with CRISPR/Cas9 vectors targeting RIPK2. Clones B7‐1 and C5‐2 were used in the study. Radioactive in vitro RIPK2 kinase assay with RIPK2 variants expressed in U2OS/NOD2 RIPK2 KO cells and purified with anti‐HA. The in vitro phosphorylated RIPK2 and universal kinase substrate MBP were separated by SDS–PAGE and exposed to X‐ray film. The inputs and precipitated proteins were analyzed by immunoblotting. Intracellular flow cytometry analysis of CXCL8 following L18‐MDP treatment (200 ng/ml, 4 h) of U2OS/NOD2 RIPK2 KO cells (clone B7‐1) reconstituted with RIPK2 variants or empty vector as indicated. Data information: Data represent the mean ± SEM of at least three independent experiments. * P

    Journal: The EMBO Journal

    Article Title: Small molecule inhibitors reveal an indispensable scaffolding role of RIPK2 in NOD2 signaling

    doi: 10.15252/embj.201899372

    Figure Lengend Snippet: RIPK2 kinase‐dead mutants support NOD2 signaling Purification of Ub‐conjugates from THP‐1 cells after treatment with L18‐MDP (200 ng/ml, 1 h) and ponatinib or GSK583 as indicated. Purified material and lysates were analyzed by immunoblotting. Schematic representation of RIPK2 mRNA and protein with indicated positions of RIPK2 gRNA sequences (CRISPR/Cas9 B and C) and epitopes for RIPK2 antibodies used in this study. E1–E11 indicates RIPK2 exons. Identification RIPK2 knockout U2OS/NOD2 cell clones grown from cell cultures transfected with CRISPR/Cas9 vectors targeting RIPK2. Clones B7‐1 and C5‐2 were used in the study. Radioactive in vitro RIPK2 kinase assay with RIPK2 variants expressed in U2OS/NOD2 RIPK2 KO cells and purified with anti‐HA. The in vitro phosphorylated RIPK2 and universal kinase substrate MBP were separated by SDS–PAGE and exposed to X‐ray film. The inputs and precipitated proteins were analyzed by immunoblotting. Intracellular flow cytometry analysis of CXCL8 following L18‐MDP treatment (200 ng/ml, 4 h) of U2OS/NOD2 RIPK2 KO cells (clone B7‐1) reconstituted with RIPK2 variants or empty vector as indicated. Data information: Data represent the mean ± SEM of at least three independent experiments. * P

    Article Snippet: Inhibitors were diluted and added in 0.5 μl DMSO 15 min prior to the addition of 1 ng/ml L18‐MDP (InvivoGen).

    Techniques: Purification, CRISPR, Knock-Out, Clone Assay, Transfection, In Vitro, Kinase Assay, SDS Page, Flow Cytometry, Cytometry, Plasmid Preparation

    Activity of select CSLP inhibitors in MDP and LPS‐stimulated RAW264.7 macrophages A, B ELISA measurement of TNF release from RAW264.7 cells treated with MDP (10 μg/ml, 24 h) (A) or LPS (10 ng/ml) (B) and CSLP compounds as indicated. Values are expressed relative to the TNF release in cells treated with MDP without inhibitor (A) or as ng/ml TNF (B). Data information: Data represent the mean ± SEM of 2–3 independent experiments. Statistical significance in (B) is determined in relation to L18‐MDP‐stimulated samples without inhibitor * P

    Journal: The EMBO Journal

    Article Title: Small molecule inhibitors reveal an indispensable scaffolding role of RIPK2 in NOD2 signaling

    doi: 10.15252/embj.201899372

    Figure Lengend Snippet: Activity of select CSLP inhibitors in MDP and LPS‐stimulated RAW264.7 macrophages A, B ELISA measurement of TNF release from RAW264.7 cells treated with MDP (10 μg/ml, 24 h) (A) or LPS (10 ng/ml) (B) and CSLP compounds as indicated. Values are expressed relative to the TNF release in cells treated with MDP without inhibitor (A) or as ng/ml TNF (B). Data information: Data represent the mean ± SEM of 2–3 independent experiments. Statistical significance in (B) is determined in relation to L18‐MDP‐stimulated samples without inhibitor * P

    Article Snippet: Inhibitors were diluted and added in 0.5 μl DMSO 15 min prior to the addition of 1 ng/ml L18‐MDP (InvivoGen).

    Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay

    Inhibition of RIPK2 ubiquitination and cIAP1 binding by CSLP inhibitors A Coomassie Blue staining of His Trap and gel filtration purified His6‐GST‐XIAP‐BIR2 WT and D214S recombinant proteins at two different concentrations as indicated. B Pulldown of RIPK2 from U2OS/NOD2 cell lysates with recombinant GST‐BIR2‐cIAP1 in the presence of CSLP inhibitors. Inhibitors were used at 100‐fold IC 50 of RIPK2 kinase activity: CSLP 37 (1.8 μM), 43 (2 μM). Purified material and lysates were analyzed by immunoblotting. C, D Purification of Ub‐conjugates from U2OS/NOD2 cells (C) or THP‐1 cells (D) after treatment with L18‐MDP (200 ng/ml, 1 h) and CSLP compounds as indicated. Purified material and lysates were analyzed by immunoblotting. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Small molecule inhibitors reveal an indispensable scaffolding role of RIPK2 in NOD2 signaling

    doi: 10.15252/embj.201899372

    Figure Lengend Snippet: Inhibition of RIPK2 ubiquitination and cIAP1 binding by CSLP inhibitors A Coomassie Blue staining of His Trap and gel filtration purified His6‐GST‐XIAP‐BIR2 WT and D214S recombinant proteins at two different concentrations as indicated. B Pulldown of RIPK2 from U2OS/NOD2 cell lysates with recombinant GST‐BIR2‐cIAP1 in the presence of CSLP inhibitors. Inhibitors were used at 100‐fold IC 50 of RIPK2 kinase activity: CSLP 37 (1.8 μM), 43 (2 μM). Purified material and lysates were analyzed by immunoblotting. C, D Purification of Ub‐conjugates from U2OS/NOD2 cells (C) or THP‐1 cells (D) after treatment with L18‐MDP (200 ng/ml, 1 h) and CSLP compounds as indicated. Purified material and lysates were analyzed by immunoblotting. Source data are available online for this figure.

    Article Snippet: Inhibitors were diluted and added in 0.5 μl DMSO 15 min prior to the addition of 1 ng/ml L18‐MDP (InvivoGen).

    Techniques: Inhibition, Binding Assay, Staining, Filtration, Purification, Recombinant, Activity Assay

    Variable sensitivity of lactobacilli to lysozyme digestion and NOD2 activation. Live or UV-killed lactobacilli were digested with lysozyme for 2 h and OD 600 assessed at t = 0 and 2h. Duplicate 2 h lysozyme digests were separated into soluble or insoluble portions via centrifugation and transfected into HEK hNOD2 cells. Positive control, L18-MDP, and negative control, Pam 2 CSK 4 , were treated similarly. (a) Data (mean with SEM) is displayed as % sensitivity to lysozyme digestion for each bacterial variant. Means with the same letter are not significantly different ( P > 0.05) from each other. (b) Data (mean with SEM) is reported as the fold increase of NFκB/AP-1 activation over negative control as determined by chemiluminescent detection of culture supernatant SEAP after 44–48 h co-culture. N = 3–8 samples per group.

    Journal: PLoS ONE

    Article Title: Nod2 is required for antigen-specific humoral responses against antigens orally delivered using a recombinant Lactobacillus vaccine platform

    doi: 10.1371/journal.pone.0196950

    Figure Lengend Snippet: Variable sensitivity of lactobacilli to lysozyme digestion and NOD2 activation. Live or UV-killed lactobacilli were digested with lysozyme for 2 h and OD 600 assessed at t = 0 and 2h. Duplicate 2 h lysozyme digests were separated into soluble or insoluble portions via centrifugation and transfected into HEK hNOD2 cells. Positive control, L18-MDP, and negative control, Pam 2 CSK 4 , were treated similarly. (a) Data (mean with SEM) is displayed as % sensitivity to lysozyme digestion for each bacterial variant. Means with the same letter are not significantly different ( P > 0.05) from each other. (b) Data (mean with SEM) is reported as the fold increase of NFκB/AP-1 activation over negative control as determined by chemiluminescent detection of culture supernatant SEAP after 44–48 h co-culture. N = 3–8 samples per group.

    Article Snippet: Positive control L18-MDP (200 ng/mL, InvivoGen) and negative controls of sterile water and Pam2 CSK4 (100 ng/mL, InvivoGen) were treated similarly.

    Techniques: Activation Assay, Centrifugation, Transfection, Positive Control, Negative Control, Variant Assay, Co-Culture Assay

    % difference of FENO after MDP challenge by baseline FENO, asthma, atopy and BHR severity. Abbreviations: FENO, fractional exhaled nitric oxide; MDP, mannitol dry powder; BHR, bronchial hyperresponsiveness; SPT, skin prick test. Baseline FENO defined as tertiles (low levels:

    Journal: PLoS ONE

    Article Title: Effect of Mannitol Dry Powder Challenge on Exhaled Nitric Oxide in Children

    doi: 10.1371/journal.pone.0054521

    Figure Lengend Snippet: % difference of FENO after MDP challenge by baseline FENO, asthma, atopy and BHR severity. Abbreviations: FENO, fractional exhaled nitric oxide; MDP, mannitol dry powder; BHR, bronchial hyperresponsiveness; SPT, skin prick test. Baseline FENO defined as tertiles (low levels:

    Article Snippet: Clinical Assessment The responsible paediatric respiratory physicians made a diagnosis (asthma, or alternative diagnoses) for every child after the first visit, taking into consideration medical history, clinical examination and all conventional test results (including bronchodilator response) before MDP challenge and FENO measurement were performed.

    Techniques: Single-particle Tracking

    Enhanced Nod1-induced cytokine production and signaling in Nod2-deficient macrophages pre-exposed to LPS and MDP. A and B , BMDMs from wild-type and Nod2 -/- mice were treated with combination of LPS and MDP or left untreated for 24 hrs. The macrophages

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Cross-tolerization between Nod1 and Nod2 Signaling Results in Reduced Refractoriness to Bacterial Infection in Nod2-Deficient Macrophages

    doi:

    Figure Lengend Snippet: Enhanced Nod1-induced cytokine production and signaling in Nod2-deficient macrophages pre-exposed to LPS and MDP. A and B , BMDMs from wild-type and Nod2 -/- mice were treated with combination of LPS and MDP or left untreated for 24 hrs. The macrophages

    Article Snippet: Ultrapure LPS from E. coli O55:B5 (Invivogen) and synthetic MDP (Bachem) were used to induce tolerization.

    Techniques: Mouse Assay

    NF-κB and MAPK activation in Nod2-deficient and wild-type macrophages infected with bacteria. A and B , BMDMs from wild-type and Nod2 -/- mice were left untreated or stimulated with combination of LPS and MDP for 24 hrs and then infected with L.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Cross-tolerization between Nod1 and Nod2 Signaling Results in Reduced Refractoriness to Bacterial Infection in Nod2-Deficient Macrophages

    doi:

    Figure Lengend Snippet: NF-κB and MAPK activation in Nod2-deficient and wild-type macrophages infected with bacteria. A and B , BMDMs from wild-type and Nod2 -/- mice were left untreated or stimulated with combination of LPS and MDP for 24 hrs and then infected with L.

    Article Snippet: Ultrapure LPS from E. coli O55:B5 (Invivogen) and synthetic MDP (Bachem) were used to induce tolerization.

    Techniques: Activation Assay, Infection, Mouse Assay

    Nod1 is required for impaired tolerization in Nod2-deficient macrophages. A and B , BMDMs from wild-type, Nod1 -/- , Nod2 -/- , and Nod1 -/- Nod2 -/- mice were stimulated with combination of LPS and MDP or left untreated for 24 hrs and then infected with live

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Cross-tolerization between Nod1 and Nod2 Signaling Results in Reduced Refractoriness to Bacterial Infection in Nod2-Deficient Macrophages

    doi:

    Figure Lengend Snippet: Nod1 is required for impaired tolerization in Nod2-deficient macrophages. A and B , BMDMs from wild-type, Nod1 -/- , Nod2 -/- , and Nod1 -/- Nod2 -/- mice were stimulated with combination of LPS and MDP or left untreated for 24 hrs and then infected with live

    Article Snippet: Ultrapure LPS from E. coli O55:B5 (Invivogen) and synthetic MDP (Bachem) were used to induce tolerization.

    Techniques: Mouse Assay, Infection

    Cross-tolerization between Nod1 and Nod2 signaling. A and B , BMDMs were left untreated (-) or pretreated with MDP or KF1B for 24 hrs to induce tolerization and then re-stimulated with MDP ( A ) or KF1B ( B ). Cell extracts were collected at the indicated

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Cross-tolerization between Nod1 and Nod2 Signaling Results in Reduced Refractoriness to Bacterial Infection in Nod2-Deficient Macrophages

    doi:

    Figure Lengend Snippet: Cross-tolerization between Nod1 and Nod2 signaling. A and B , BMDMs were left untreated (-) or pretreated with MDP or KF1B for 24 hrs to induce tolerization and then re-stimulated with MDP ( A ) or KF1B ( B ). Cell extracts were collected at the indicated

    Article Snippet: Ultrapure LPS from E. coli O55:B5 (Invivogen) and synthetic MDP (Bachem) were used to induce tolerization.

    Techniques:

    Pre-exposure to Nod1 ligand inhibits bacterial-induced cytokine responses in Nod2-deficient macrophages stimulated with LPS and MDP. A-D , BMDMs from wild-type and Nod2 -/- mice were left untreated or stimulated with combination of LPS and MDP or combination

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Cross-tolerization between Nod1 and Nod2 Signaling Results in Reduced Refractoriness to Bacterial Infection in Nod2-Deficient Macrophages

    doi:

    Figure Lengend Snippet: Pre-exposure to Nod1 ligand inhibits bacterial-induced cytokine responses in Nod2-deficient macrophages stimulated with LPS and MDP. A-D , BMDMs from wild-type and Nod2 -/- mice were left untreated or stimulated with combination of LPS and MDP or combination

    Article Snippet: Ultrapure LPS from E. coli O55:B5 (Invivogen) and synthetic MDP (Bachem) were used to induce tolerization.

    Techniques: Mouse Assay

    Nod2 deficiency is associated with reduced tolerization to bacterial infection in macrophages pre-exposed to LPS and MDP. A-D , BMDMs from wild-type and Nod2 -/- mice were stimulated with LPS+MDP or left untreated for 24 hrs and then infected with live

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Cross-tolerization between Nod1 and Nod2 Signaling Results in Reduced Refractoriness to Bacterial Infection in Nod2-Deficient Macrophages

    doi:

    Figure Lengend Snippet: Nod2 deficiency is associated with reduced tolerization to bacterial infection in macrophages pre-exposed to LPS and MDP. A-D , BMDMs from wild-type and Nod2 -/- mice were stimulated with LPS+MDP or left untreated for 24 hrs and then infected with live

    Article Snippet: Ultrapure LPS from E. coli O55:B5 (Invivogen) and synthetic MDP (Bachem) were used to induce tolerization.

    Techniques: Infection, Mouse Assay

    GSK669 exhibits NOD2 selectivity in cells that express endogenous NOD1 and NOD2. Concentration response curves of GSK669 and its close analogue GSK400 for the inhibition of MDP (A) but not Tri-DAP (B) stimulated IL-8 secretion in HCT116 cells which express functionally active NOD1 and NOD2. Cells were pre-incubated with compounds for 1 hour prior to addition of NOD agonists. IL-8 secreted into medium was assayed after 24 hours. The same RIP2 inhibitor used in Figure 2C was included as a positive control. Data are the average percent inhibition obtained from 1 (RIP2), 2 (GSK400) or 4 (GSK669) separate experiments.

    Journal: PLoS ONE

    Article Title: Identification of Benzimidazole Diamides as Selective Inhibitors of the Nucleotide-Binding Oligomerization Domain 2 (NOD2) Signaling Pathway

    doi: 10.1371/journal.pone.0069619

    Figure Lengend Snippet: GSK669 exhibits NOD2 selectivity in cells that express endogenous NOD1 and NOD2. Concentration response curves of GSK669 and its close analogue GSK400 for the inhibition of MDP (A) but not Tri-DAP (B) stimulated IL-8 secretion in HCT116 cells which express functionally active NOD1 and NOD2. Cells were pre-incubated with compounds for 1 hour prior to addition of NOD agonists. IL-8 secreted into medium was assayed after 24 hours. The same RIP2 inhibitor used in Figure 2C was included as a positive control. Data are the average percent inhibition obtained from 1 (RIP2), 2 (GSK400) or 4 (GSK669) separate experiments.

    Article Snippet: The NOD activators iE-DAP, Tri-DAP, MDP, MDP-rhodamine, the TLR2/6 ligand Pam2 CSK4, single stranded RNA (ssRNA40) and the antibiotic Blasticidin S used for maintenance culture of stable cell lines, were all obtained from InvivoGen.

    Techniques: Concentration Assay, Inhibition, Incubation, Positive Control

    GSK669 inhibits NOD2 but not NOD1 mediated responses. (A) Structure of GSK669, the original hit identified from the NOD2 HTS screening cascade. (B) Concentration response curves showing GSK669 selectively inhibits MDP-stimulated IL-8. IL-8 secretion in either MDP treated 293/hNOD2 or iE-DAP treated 293/hNOD1 stable cell lines was measured in the presence of increasing concentrations of GSK669. Data are mean ± SD from 6 independent assays. (C) Selective inhibition of NOD2-mediated MAPK phosphorylation by GSK669. Serum-starved 293/hNOD1 or NOD2 stable cells were pre-incubated with compound at the concentrations indicated and then stimulated for 1 hour with either Tri-DAP or MDP, respectively. Phospho-p38, JNK and ERK1/2 were identified in cell lysates by western blotting. A RIP2 inhibitor compound was used as a positive control. Similar results were obtained in 5 (293/hNOD2) and 2 (293/hNOD1) separate experiments.

    Journal: PLoS ONE

    Article Title: Identification of Benzimidazole Diamides as Selective Inhibitors of the Nucleotide-Binding Oligomerization Domain 2 (NOD2) Signaling Pathway

    doi: 10.1371/journal.pone.0069619

    Figure Lengend Snippet: GSK669 inhibits NOD2 but not NOD1 mediated responses. (A) Structure of GSK669, the original hit identified from the NOD2 HTS screening cascade. (B) Concentration response curves showing GSK669 selectively inhibits MDP-stimulated IL-8. IL-8 secretion in either MDP treated 293/hNOD2 or iE-DAP treated 293/hNOD1 stable cell lines was measured in the presence of increasing concentrations of GSK669. Data are mean ± SD from 6 independent assays. (C) Selective inhibition of NOD2-mediated MAPK phosphorylation by GSK669. Serum-starved 293/hNOD1 or NOD2 stable cells were pre-incubated with compound at the concentrations indicated and then stimulated for 1 hour with either Tri-DAP or MDP, respectively. Phospho-p38, JNK and ERK1/2 were identified in cell lysates by western blotting. A RIP2 inhibitor compound was used as a positive control. Similar results were obtained in 5 (293/hNOD2) and 2 (293/hNOD1) separate experiments.

    Article Snippet: The NOD activators iE-DAP, Tri-DAP, MDP, MDP-rhodamine, the TLR2/6 ligand Pam2 CSK4, single stranded RNA (ssRNA40) and the antibiotic Blasticidin S used for maintenance culture of stable cell lines, were all obtained from InvivoGen.

    Techniques: Concentration Assay, Stable Transfection, Inhibition, Incubation, Western Blot, Positive Control

    GSK669 and its analog GSK717 suppress cytokine secretion by primary human monocytes. NOD2 inhibitors suppress cytokine secretion in primary human monocytes activated via NOD2 alone as well as via synergistic NOD2-TLR2 co-activation. (A) Monocytes isolated from whole blood were immediately pre-incubated with NOD2 inhibitor compounds (5 μM) for 1 hour and then stimulated with MDP (0.1 μg/mL) for 24 hours. The concentration of IL-1β, IL-6, IL-8 and TNFα in the conditioned medium was determined. Data are mean ± SD of triplicate treatments from a representative experiment repeated once with similar results. (B) GSK717 blocks synergy between NOD2 and TLR2. Primary human monocytes were pre-incubated for 1 hour with either RIP2 inhibitor alone (0.05–1.6 μM), GSK717 alone (0.5–16 μM) or the combination of GSK717 (0.5–16 μM) and RIP2 inhibitor (at a fixed concentration of 1.6 μM) and then stimulated with MDP (0.1 μg/mL), Pam 2 CSK4 (100 pg/mL) or both agonists. IL-8 secreted into the medium was determined after 24 hours. The dotted line indicates the level of IL-8 released by co-stimulated cells treated with the highest concentration of RIP2 inhibitor. Results are representative of three independent experiments.

    Journal: PLoS ONE

    Article Title: Identification of Benzimidazole Diamides as Selective Inhibitors of the Nucleotide-Binding Oligomerization Domain 2 (NOD2) Signaling Pathway

    doi: 10.1371/journal.pone.0069619

    Figure Lengend Snippet: GSK669 and its analog GSK717 suppress cytokine secretion by primary human monocytes. NOD2 inhibitors suppress cytokine secretion in primary human monocytes activated via NOD2 alone as well as via synergistic NOD2-TLR2 co-activation. (A) Monocytes isolated from whole blood were immediately pre-incubated with NOD2 inhibitor compounds (5 μM) for 1 hour and then stimulated with MDP (0.1 μg/mL) for 24 hours. The concentration of IL-1β, IL-6, IL-8 and TNFα in the conditioned medium was determined. Data are mean ± SD of triplicate treatments from a representative experiment repeated once with similar results. (B) GSK717 blocks synergy between NOD2 and TLR2. Primary human monocytes were pre-incubated for 1 hour with either RIP2 inhibitor alone (0.05–1.6 μM), GSK717 alone (0.5–16 μM) or the combination of GSK717 (0.5–16 μM) and RIP2 inhibitor (at a fixed concentration of 1.6 μM) and then stimulated with MDP (0.1 μg/mL), Pam 2 CSK4 (100 pg/mL) or both agonists. IL-8 secreted into the medium was determined after 24 hours. The dotted line indicates the level of IL-8 released by co-stimulated cells treated with the highest concentration of RIP2 inhibitor. Results are representative of three independent experiments.

    Article Snippet: The NOD activators iE-DAP, Tri-DAP, MDP, MDP-rhodamine, the TLR2/6 ligand Pam2 CSK4, single stranded RNA (ssRNA40) and the antibiotic Blasticidin S used for maintenance culture of stable cell lines, were all obtained from InvivoGen.

    Techniques: Activation Assay, Isolation, Incubation, Concentration Assay

    Cell-based screening scheme used to identify selective NOD2 inhibitors. The compound collection was screened for inhibitors of MDP-induced IL-8 secretion in 293/hNOD2 stable cells. Active inhibitors were then tested for selectivity against IL-8 induced by alternative pathways including NOD1, TNFR1 and TLR2 as well as for direct inhibitors of RIP2 kinase. The activity of selective NOD2 inhibitors was then confirmed in cells expressing NOD2 endogenously and against agonist-independent NOD2 signaling.

    Journal: PLoS ONE

    Article Title: Identification of Benzimidazole Diamides as Selective Inhibitors of the Nucleotide-Binding Oligomerization Domain 2 (NOD2) Signaling Pathway

    doi: 10.1371/journal.pone.0069619

    Figure Lengend Snippet: Cell-based screening scheme used to identify selective NOD2 inhibitors. The compound collection was screened for inhibitors of MDP-induced IL-8 secretion in 293/hNOD2 stable cells. Active inhibitors were then tested for selectivity against IL-8 induced by alternative pathways including NOD1, TNFR1 and TLR2 as well as for direct inhibitors of RIP2 kinase. The activity of selective NOD2 inhibitors was then confirmed in cells expressing NOD2 endogenously and against agonist-independent NOD2 signaling.

    Article Snippet: The NOD activators iE-DAP, Tri-DAP, MDP, MDP-rhodamine, the TLR2/6 ligand Pam2 CSK4, single stranded RNA (ssRNA40) and the antibiotic Blasticidin S used for maintenance culture of stable cell lines, were all obtained from InvivoGen.

    Techniques: Activity Assay, Expressing

    The activity of GSK669 is specific for inhibition of MDP-stimulated NOD2 responses. (A and B) Over-expression of NOD2 by viral transduction in HEK293T cells induces secretion of IL-8. Cells were infected (MOI = 50) with NOD1 or NOD2 bacmam virus alone for either 24 hours (A) or for 6 hours followed by removal of virus and stimulation with iE-DAP (300 ng/mL) or MDP (30 ng/mL) for an additional 24 hours (B). GSK669 (5 μM) failed to block IL-8 secretion induced by NOD1/2 virus alone but selectively blocked the MDP response in NOD2 transduced cells. The IKK inhibitor (0.5 μM) suppressed IL-8 release under all conditions. Data in (A and B) are the mean ± SD and are representative of two experiments. (C and D) Liposome-mediated transfection of ssRNA dose-dependently induces IFNβ gene expression in 293/hNOD2 but not 293/hNOD1 stable cells and the response was unaffected by GSK669 (0.16–16 μM) and a RIP2 inhibitor (0.1 μM). Cells were pre-incubated with compound for 30 minutes prior to transfection with ssRNA. IFNβ mRNA levels were measured after 6 hours by real-time RT-PCR. Data are presented as fold increase over mock transfected cells (mean ± SD) and are representative of two independent experiments.

    Journal: PLoS ONE

    Article Title: Identification of Benzimidazole Diamides as Selective Inhibitors of the Nucleotide-Binding Oligomerization Domain 2 (NOD2) Signaling Pathway

    doi: 10.1371/journal.pone.0069619

    Figure Lengend Snippet: The activity of GSK669 is specific for inhibition of MDP-stimulated NOD2 responses. (A and B) Over-expression of NOD2 by viral transduction in HEK293T cells induces secretion of IL-8. Cells were infected (MOI = 50) with NOD1 or NOD2 bacmam virus alone for either 24 hours (A) or for 6 hours followed by removal of virus and stimulation with iE-DAP (300 ng/mL) or MDP (30 ng/mL) for an additional 24 hours (B). GSK669 (5 μM) failed to block IL-8 secretion induced by NOD1/2 virus alone but selectively blocked the MDP response in NOD2 transduced cells. The IKK inhibitor (0.5 μM) suppressed IL-8 release under all conditions. Data in (A and B) are the mean ± SD and are representative of two experiments. (C and D) Liposome-mediated transfection of ssRNA dose-dependently induces IFNβ gene expression in 293/hNOD2 but not 293/hNOD1 stable cells and the response was unaffected by GSK669 (0.16–16 μM) and a RIP2 inhibitor (0.1 μM). Cells were pre-incubated with compound for 30 minutes prior to transfection with ssRNA. IFNβ mRNA levels were measured after 6 hours by real-time RT-PCR. Data are presented as fold increase over mock transfected cells (mean ± SD) and are representative of two independent experiments.

    Article Snippet: The NOD activators iE-DAP, Tri-DAP, MDP, MDP-rhodamine, the TLR2/6 ligand Pam2 CSK4, single stranded RNA (ssRNA40) and the antibiotic Blasticidin S used for maintenance culture of stable cell lines, were all obtained from InvivoGen.

    Techniques: Activity Assay, Inhibition, Over Expression, Transduction, Infection, Blocking Assay, Transfection, Expressing, Incubation, Quantitative RT-PCR

    ROC curve analysis between the histologic response and each parameter of the 18 F-FDG PET and 99m Tc-MDP bone scans. In ROC curves using the 18 F-FDG PET parameters (A), Δ%SUV max shows the largest AUC value (0.829). The AUC values for SUV max 1 and SUV max 2 are 0.571 and 0.817, respectively. In ROC curves using 99m Tc-MDP bone scan parameters (B), the AUC for Δ%T/NT max has the largest value (0.772). The AUC values for T/NT max 1 and T/NT max 2 are 0.601 and 0.770, respectively. The ROC curves regarding Δ%SUV max and Δ%T/NT max are compared (C). Two curves did not show significant difference ( P = 0.44). 18 F-FDG PET = 18 F-Fluorodeoxyglucose positron emission tomography, 99m Tc-MDP = 99m Tc-methyl diphosphonate, Δ%SUV max = percent changes of the maximum standardized uptake value, Δ%T/NT max = percent changes of the maximum tumor-to-nontumor ratio, AUC = area under the curve, ROC = receiver operating characteristic.

    Journal: Medicine

    Article Title: Comparison of 99mTc-methyl diphosphonate bone scintigraphy and 18F-fluorodeoxyglucose positron emission tomography/computed tomography to predict histologic response to neoadjuvant chemotherapy in patients with osteosarcoma

    doi: 10.1097/MD.0000000000012318

    Figure Lengend Snippet: ROC curve analysis between the histologic response and each parameter of the 18 F-FDG PET and 99m Tc-MDP bone scans. In ROC curves using the 18 F-FDG PET parameters (A), Δ%SUV max shows the largest AUC value (0.829). The AUC values for SUV max 1 and SUV max 2 are 0.571 and 0.817, respectively. In ROC curves using 99m Tc-MDP bone scan parameters (B), the AUC for Δ%T/NT max has the largest value (0.772). The AUC values for T/NT max 1 and T/NT max 2 are 0.601 and 0.770, respectively. The ROC curves regarding Δ%SUV max and Δ%T/NT max are compared (C). Two curves did not show significant difference ( P = 0.44). 18 F-FDG PET = 18 F-Fluorodeoxyglucose positron emission tomography, 99m Tc-MDP = 99m Tc-methyl diphosphonate, Δ%SUV max = percent changes of the maximum standardized uptake value, Δ%T/NT max = percent changes of the maximum tumor-to-nontumor ratio, AUC = area under the curve, ROC = receiver operating characteristic.

    Article Snippet: Bone scan using 99m Tc-MDP is a sensitive tool for detecting primary bone tumor and bone metastasis,[ ] but it has limitations in reflecting the treatment response in a relatively short time.

    Techniques: Positron Emission Tomography

    iE-DAP, MDP and Poly(I:C)LyoVec induce T-cell proliferation

    Journal: Immunology

    Article Title: Nucleotide-binding and oligomerization domain-like receptors and retinoic acid inducible gene-like receptors in human tonsillar T lymphocytes

    doi: 10.1111/j.1365-2567.2011.03414.x

    Figure Lengend Snippet: iE-DAP, MDP and Poly(I:C)LyoVec induce T-cell proliferation

    Article Snippet: The following reagents were used: iE-DAP, d -glutamyl lysine (iE-Lys), MDP and MDP control (endotoxin levels < 0·125 EU/ml) together with Poly(I:C)/LyoVec from Invivogen (San Diego, CA).

    Techniques:

    NLRP12 suppresses MDP-induced NF-κB activation by targeting the NOD2/RIPK2 complex for assembling of K48 poly-ubiquitin chains on NOD2 and subsequent degradation. a Western blot analysis of NOD2 ubiquitination in HEK293T cells that were transfected with FLAG-tagged NOD2 in the presence of increasing amounts of NLRP12 as indicated. b Western blot analysis of NOD2 stability in the presence of cycloheximide (CHX at 20 μg/mL) for 4 h. HEK293T cell extracts were subjected to western blot analysis for FLAG-tagged NOD2, Myc-tagged NLRP12 and β-actin. c NF-κB-Luciferase activity in HEK293T cells normalized to expression of β-galactosidase. Depicted are mean ± SEM ( n = 3). d Immunoblot analysis of poly-K48 ubiquitination on NOD2 and of HSP90α/β following co-immunoprecipitation with NOD2 by using the anti-Myc antibody in THP-1 Myc-BirA*-NOD2 cells that were stimulated with MDP (10 µg/mL; left panel) or LPS (0.2 µg/mL, right panel) for 2 h with or without MG132 (12.5 µM)

    Journal: Nature Communications

    Article Title: Proteasomal degradation of NOD2 by NLRP12 in monocytes promotes bacterial tolerance and colonization by enteropathogens

    doi: 10.1038/s41467-018-07750-5

    Figure Lengend Snippet: NLRP12 suppresses MDP-induced NF-κB activation by targeting the NOD2/RIPK2 complex for assembling of K48 poly-ubiquitin chains on NOD2 and subsequent degradation. a Western blot analysis of NOD2 ubiquitination in HEK293T cells that were transfected with FLAG-tagged NOD2 in the presence of increasing amounts of NLRP12 as indicated. b Western blot analysis of NOD2 stability in the presence of cycloheximide (CHX at 20 μg/mL) for 4 h. HEK293T cell extracts were subjected to western blot analysis for FLAG-tagged NOD2, Myc-tagged NLRP12 and β-actin. c NF-κB-Luciferase activity in HEK293T cells normalized to expression of β-galactosidase. Depicted are mean ± SEM ( n = 3). d Immunoblot analysis of poly-K48 ubiquitination on NOD2 and of HSP90α/β following co-immunoprecipitation with NOD2 by using the anti-Myc antibody in THP-1 Myc-BirA*-NOD2 cells that were stimulated with MDP (10 µg/mL; left panel) or LPS (0.2 µg/mL, right panel) for 2 h with or without MG132 (12.5 µM)

    Article Snippet: Fresh cells were stimulated with Ultrapure LPS (10 ng/mL; Sigma-Aldrich), MDP (10–100 µg/mL; Invivogen), MDP-DD (10–100 µg/mL; Invivogen), or left unstimulated for 24 h. Supernatants were collected for ELISA analysis.

    Techniques: Activation Assay, Western Blot, Transfection, Luciferase, Activity Assay, Expressing, Immunoprecipitation

    LPS-primed Nlrp12 -deficient mice are highly susceptible to secondary challenge by bacterial MDP. a The survival of wild-type mice ( n = 8) and Nlrp12 −/− mice ( n = 7) was plotted after i.p. administration of a lethal dose of ultrapure LPS from E. coli 0111:B4 (54 mg/kg). b The survival of wild-type mice ( n = 18) and Nlrp12 -deficient mice ( n = 9) was plotted after a non-lethal dose of ultrapure LPS from E. coli 0111:B4 (10 mg/kg) that was followed 24 h later with i.p. Murabutide administration at 10 mg/kg. c The survival of wild-type mice ( n = 8), Nlrp12 −/− mice ( n = 7) and compound mutant mice ( n = 8) was plotted after a non-lethal dose of ultrapure LPS from E. coli 0111:B4 (2 mg/kg) that was followed 24 h later with i.p. Murabutide administration at 10 mg/kg. P -value by log-rank test

    Journal: Nature Communications

    Article Title: Proteasomal degradation of NOD2 by NLRP12 in monocytes promotes bacterial tolerance and colonization by enteropathogens

    doi: 10.1038/s41467-018-07750-5

    Figure Lengend Snippet: LPS-primed Nlrp12 -deficient mice are highly susceptible to secondary challenge by bacterial MDP. a The survival of wild-type mice ( n = 8) and Nlrp12 −/− mice ( n = 7) was plotted after i.p. administration of a lethal dose of ultrapure LPS from E. coli 0111:B4 (54 mg/kg). b The survival of wild-type mice ( n = 18) and Nlrp12 -deficient mice ( n = 9) was plotted after a non-lethal dose of ultrapure LPS from E. coli 0111:B4 (10 mg/kg) that was followed 24 h later with i.p. Murabutide administration at 10 mg/kg. c The survival of wild-type mice ( n = 8), Nlrp12 −/− mice ( n = 7) and compound mutant mice ( n = 8) was plotted after a non-lethal dose of ultrapure LPS from E. coli 0111:B4 (2 mg/kg) that was followed 24 h later with i.p. Murabutide administration at 10 mg/kg. P -value by log-rank test

    Article Snippet: Fresh cells were stimulated with Ultrapure LPS (10 ng/mL; Sigma-Aldrich), MDP (10–100 µg/mL; Invivogen), MDP-DD (10–100 µg/mL; Invivogen), or left unstimulated for 24 h. Supernatants were collected for ELISA analysis.

    Techniques: Mouse Assay, Mutagenesis