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  • 97
    Name:
    IL 1β D4T2D Rabbit mAb Mouse Specific
    Description:
    Interleukin 1β IL 1β is a proinflammatory cytokine produced predominantly by activated monocytes and epithelial cells 1 Precursor IL 1β is cleaved by caspase 1 and mature IL 1β is then secreted 1 3 Target cells include macrophages and many other cell types Signaling by IL 1β involves IL 1β binding to IL 1 accessory protein IL 1 AcP the complex then binds to IL 1RI 1 2 Signaling occurs through activation of MAP kinase and NF κB pathways 1 2 IL 1β also binds to IL 1RII which lacks an intracellular signaling domain and thereby serves as a high affinity decoy receptor IL 1β binding to IL 1RI is inhibited by the negative regulator IL 1R antagonist IL 1Ra IL 1Ra binding to IL 1RI does not signal and serves to block IL 1β signaling IL 1β plays critical roles in the acute phase response and sepsis 1 3
    Catalog Number:
    12426
    Price:
    None
    Category:
    Primary Antibodies
    Source:
    Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val175 of mouse IL-1β protein.
    Reactivity:
    Mouse
    Applications:
    Western Blot
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc il 1β
    Pre‐ and post‐treatment with CAT639 protect INS1E beta cells. (A, C) Pretreatment with CAT639 for 6 h decreased caspase‐9 activity induced by cytokines (upper panels) and attenuated the impaired cell viability induced by cytokines (bottom panels). INS1E beta cells were pretreated with compounds for 6 h, followed by co‐treatment with cytokines (10 ng·mL −1 <t>IL‐1β</t> and 100 ng·mL −1 IFNγ) for an additional 24 h, and caspase‐9 and cell viability were measured by caspase Glo‐9 or CTG, according to the manufacturer's instructions ( n = 6). (B, D) Post‐treatment of CAT639 6 h after cytokine treatment also partially decreased caspase‐9 activity induced by cytokines (upper panels) and restored cell viability impaired by cytokines (bottom panels). INS1E beta cells were plated overnight and treated with cytokines (10 ng·mL −1 IL‐1β and 100 ng·mL −1 IFNγ) for 6 h followed by compound treatment for 24 h, and caspase‐9 and cell viability were measured by caspase Glo‐9 or CTG, according to the manufacturer's manual ( n = 6). Data are presented as mean ± SD. ** P
    Interleukin 1β IL 1β is a proinflammatory cytokine produced predominantly by activated monocytes and epithelial cells 1 Precursor IL 1β is cleaved by caspase 1 and mature IL 1β is then secreted 1 3 Target cells include macrophages and many other cell types Signaling by IL 1β involves IL 1β binding to IL 1 accessory protein IL 1 AcP the complex then binds to IL 1RI 1 2 Signaling occurs through activation of MAP kinase and NF κB pathways 1 2 IL 1β also binds to IL 1RII which lacks an intracellular signaling domain and thereby serves as a high affinity decoy receptor IL 1β binding to IL 1RI is inhibited by the negative regulator IL 1R antagonist IL 1Ra IL 1Ra binding to IL 1RI does not signal and serves to block IL 1β signaling IL 1β plays critical roles in the acute phase response and sepsis 1 3
    https://www.bioz.com/result/il 1β/product/Cell Signaling Technology Inc
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    il 1β - by Bioz Stars, 2021-05
    97/100 stars

    Images

    1) Product Images from "A novel inhibitor of inducible NOS dimerization protects against cytokine‐induced rat beta cell dysfunction"

    Article Title: A novel inhibitor of inducible NOS dimerization protects against cytokine‐induced rat beta cell dysfunction

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.14388

    Pre‐ and post‐treatment with CAT639 protect INS1E beta cells. (A, C) Pretreatment with CAT639 for 6 h decreased caspase‐9 activity induced by cytokines (upper panels) and attenuated the impaired cell viability induced by cytokines (bottom panels). INS1E beta cells were pretreated with compounds for 6 h, followed by co‐treatment with cytokines (10 ng·mL −1 IL‐1β and 100 ng·mL −1 IFNγ) for an additional 24 h, and caspase‐9 and cell viability were measured by caspase Glo‐9 or CTG, according to the manufacturer's instructions ( n = 6). (B, D) Post‐treatment of CAT639 6 h after cytokine treatment also partially decreased caspase‐9 activity induced by cytokines (upper panels) and restored cell viability impaired by cytokines (bottom panels). INS1E beta cells were plated overnight and treated with cytokines (10 ng·mL −1 IL‐1β and 100 ng·mL −1 IFNγ) for 6 h followed by compound treatment for 24 h, and caspase‐9 and cell viability were measured by caspase Glo‐9 or CTG, according to the manufacturer's manual ( n = 6). Data are presented as mean ± SD. ** P
    Figure Legend Snippet: Pre‐ and post‐treatment with CAT639 protect INS1E beta cells. (A, C) Pretreatment with CAT639 for 6 h decreased caspase‐9 activity induced by cytokines (upper panels) and attenuated the impaired cell viability induced by cytokines (bottom panels). INS1E beta cells were pretreated with compounds for 6 h, followed by co‐treatment with cytokines (10 ng·mL −1 IL‐1β and 100 ng·mL −1 IFNγ) for an additional 24 h, and caspase‐9 and cell viability were measured by caspase Glo‐9 or CTG, according to the manufacturer's instructions ( n = 6). (B, D) Post‐treatment of CAT639 6 h after cytokine treatment also partially decreased caspase‐9 activity induced by cytokines (upper panels) and restored cell viability impaired by cytokines (bottom panels). INS1E beta cells were plated overnight and treated with cytokines (10 ng·mL −1 IL‐1β and 100 ng·mL −1 IFNγ) for 6 h followed by compound treatment for 24 h, and caspase‐9 and cell viability were measured by caspase Glo‐9 or CTG, according to the manufacturer's manual ( n = 6). Data are presented as mean ± SD. ** P

    Techniques Used: Activity Assay, CTG Assay

    High throughput screening for small molecule inhibitors of mitochondrial apoptosis. (A) A schematic description of the intrinsic (mitochondrial) pathway of apoptosis. Cytokines induce intracellular stress and ultimately apoptosis. A high‐content image‐based phenotypic assay was designed to identify small molecules that protect beta cells from cytokine‐induced activation of mitochondrial apoptosis by monitoring cleaved caspase‐9 levels. (B) Representative screening set‐up from a subset of Life Chemicals Library (30 000 compounds), which identified the hit ATV399. Rat insulin‐producing INS1E cells were pretreated with compounds for 24 h. Cytokines (2.5 ng·mL −1 IL‐1β and 100 ng·mL −1 IFNγ) were then supplemented in the presence of high glucose (33 mM) to induce intracellular stress and activate cleaved caspase‐9. Compounds reducing cleaved caspase‐9 levels by > 50% in the presence of cytokines were considered as hits. Z' > 0.7 was calculated based on DMSO versus cytokine‐treated wells, indicating that the assay was robust and suitable for high throughput screening. ATV399 was identified as one of the most potent hit compounds. (C) Schematic workflow of the high‐throughput screen. (D) Chemical structures of the selected hit compounds. Everolimus, IKK‐16, baricitinib and flumazenil were identified as hit compounds with known mechanisms of action.
    Figure Legend Snippet: High throughput screening for small molecule inhibitors of mitochondrial apoptosis. (A) A schematic description of the intrinsic (mitochondrial) pathway of apoptosis. Cytokines induce intracellular stress and ultimately apoptosis. A high‐content image‐based phenotypic assay was designed to identify small molecules that protect beta cells from cytokine‐induced activation of mitochondrial apoptosis by monitoring cleaved caspase‐9 levels. (B) Representative screening set‐up from a subset of Life Chemicals Library (30 000 compounds), which identified the hit ATV399. Rat insulin‐producing INS1E cells were pretreated with compounds for 24 h. Cytokines (2.5 ng·mL −1 IL‐1β and 100 ng·mL −1 IFNγ) were then supplemented in the presence of high glucose (33 mM) to induce intracellular stress and activate cleaved caspase‐9. Compounds reducing cleaved caspase‐9 levels by > 50% in the presence of cytokines were considered as hits. Z' > 0.7 was calculated based on DMSO versus cytokine‐treated wells, indicating that the assay was robust and suitable for high throughput screening. ATV399 was identified as one of the most potent hit compounds. (C) Schematic workflow of the high‐throughput screen. (D) Chemical structures of the selected hit compounds. Everolimus, IKK‐16, baricitinib and flumazenil were identified as hit compounds with known mechanisms of action.

    Techniques Used: High Throughput Screening Assay, Phenotypic Assay, Activation Assay

    CAT639 inhibited dimerization of iNOS. (A) CAT639 slightly inhibited iNOS mRNA expression ( n = 3). (B) CAT639 inhibited NO production induced by IL‐1 alone or IL‐1β + IFN‐γ. NO was measured using a nitrate/nitrite colorimetric assay kit ( n = 3). (C) CAT639 had no effect on apoptosis induced by SNAP ( n = 3). (D) CAT639 destabilized iNOS dimer in rat insulin‐producing INS1E cells. Dimeric proteins were determined by performing low‐temperature SDS‐PAGE. Samples lysed in sample buffer with β‐ME and boiled for 5 min were used as a positive control of total iNOS. Data are presented as mean ± SD.
    Figure Legend Snippet: CAT639 inhibited dimerization of iNOS. (A) CAT639 slightly inhibited iNOS mRNA expression ( n = 3). (B) CAT639 inhibited NO production induced by IL‐1 alone or IL‐1β + IFN‐γ. NO was measured using a nitrate/nitrite colorimetric assay kit ( n = 3). (C) CAT639 had no effect on apoptosis induced by SNAP ( n = 3). (D) CAT639 destabilized iNOS dimer in rat insulin‐producing INS1E cells. Dimeric proteins were determined by performing low‐temperature SDS‐PAGE. Samples lysed in sample buffer with β‐ME and boiled for 5 min were used as a positive control of total iNOS. Data are presented as mean ± SD.

    Techniques Used: Expressing, Nitrate Nitrite Colorimetric Assay, SDS Page, Positive Control

    2) Product Images from "Baicalin Inhibits NOD-Like Receptor Family, Pyrin Containing Domain 3 Inflammasome Activation in Murine Macrophages by Augmenting Protein Kinase A Signaling"

    Article Title: Baicalin Inhibits NOD-Like Receptor Family, Pyrin Containing Domain 3 Inflammasome Activation in Murine Macrophages by Augmenting Protein Kinase A Signaling

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01409

    Baicalin inhibited cell death in macrophages depending on protein kinase A (PKA) signaling. LPS-primed bone marrow-derived macrophages were pretreated with the PKA inhibitor H89 (20 µM) for 30 min and then incubated with graded doses of baicalin for 1 h, followed by stimulated with adenosine triphosphate (ATP) (3 mM) for 30 min. (A) Cell death was assay by propidium iodide (PI) (red) and Hoechst 33342 (blue) co-staining for 10 min. The images were captured by fluorescence microscopy. One set of representative images of three independent experiments is shown. Scale bars, 50 µm. (B) PI-positive cells in five randomly chosen fields each containing ~100 cells were quantified. The percentage of cell death is defined as the ratio of PI-positive cells relative to all cells (revealed by Hoechst). Data are shown as mean ± SD ( n = 5). (C) Cells were treated as in panel (A) . The levels of soluble interleukin (IL)-1β in culture supernatants were measured by cytometric bead array assay. Data are shown as mean ± SD ( n = 3). * P
    Figure Legend Snippet: Baicalin inhibited cell death in macrophages depending on protein kinase A (PKA) signaling. LPS-primed bone marrow-derived macrophages were pretreated with the PKA inhibitor H89 (20 µM) for 30 min and then incubated with graded doses of baicalin for 1 h, followed by stimulated with adenosine triphosphate (ATP) (3 mM) for 30 min. (A) Cell death was assay by propidium iodide (PI) (red) and Hoechst 33342 (blue) co-staining for 10 min. The images were captured by fluorescence microscopy. One set of representative images of three independent experiments is shown. Scale bars, 50 µm. (B) PI-positive cells in five randomly chosen fields each containing ~100 cells were quantified. The percentage of cell death is defined as the ratio of PI-positive cells relative to all cells (revealed by Hoechst). Data are shown as mean ± SD ( n = 5). (C) Cells were treated as in panel (A) . The levels of soluble interleukin (IL)-1β in culture supernatants were measured by cytometric bead array assay. Data are shown as mean ± SD ( n = 3). * P

    Techniques Used: Derivative Assay, Incubation, Staining, Fluorescence, Microscopy

    Baicalin inhibited NOD-like receptor (NLR) family, pyrin containing domain 3 (NLRP3) inflammasome activation depending on the adenyl cyclase activity. Bone marrow-derived macrophages were primed with LPS (500 ng/ml) for 4 h, pretreated with baicalin (0.3 mM) for 30 min, and then incubated with adenyl cyclase inhibitor MDL12330A (10 µM) for 30 min, followed by co-treatment with adenosine triphosphate (ATP) (3 mM) for 30 min. (A) Western blot analysis was used to detect the expression and secretion of interleukin (IL)-1β in the cell lysates and culture supernatants, respectively. β-Tubulin was used as a loading control for cell lysates. (B) . Histograms showing the relative intensity of mature IL-1β band in panel (A) ( n = 3). The intensity of IL-1β band in ATP group was set to 1.0 while those of the other groups were calculated relative to the ATP group. (C) Cell death was measured by staining with propidium iodide (PI) (red, staining dead cells) and Hoechst 33342 (blue, staining all nuclei) together for 10 min. All images were captured by fluorescence microscopy and showed in merge with bright-field images. One set of representative images of three independent experiments is shown. Scale bars, 50 µm. (D) PI-positive cells in five randomly chosen fields each containing ~100 cells were quantified. The percentage of cell death is defined as the ratio of PI-positive cells relative to all cells (revealed by Hoechst). Data are shown as mean ± SD ( n = 5). * P
    Figure Legend Snippet: Baicalin inhibited NOD-like receptor (NLR) family, pyrin containing domain 3 (NLRP3) inflammasome activation depending on the adenyl cyclase activity. Bone marrow-derived macrophages were primed with LPS (500 ng/ml) for 4 h, pretreated with baicalin (0.3 mM) for 30 min, and then incubated with adenyl cyclase inhibitor MDL12330A (10 µM) for 30 min, followed by co-treatment with adenosine triphosphate (ATP) (3 mM) for 30 min. (A) Western blot analysis was used to detect the expression and secretion of interleukin (IL)-1β in the cell lysates and culture supernatants, respectively. β-Tubulin was used as a loading control for cell lysates. (B) . Histograms showing the relative intensity of mature IL-1β band in panel (A) ( n = 3). The intensity of IL-1β band in ATP group was set to 1.0 while those of the other groups were calculated relative to the ATP group. (C) Cell death was measured by staining with propidium iodide (PI) (red, staining dead cells) and Hoechst 33342 (blue, staining all nuclei) together for 10 min. All images were captured by fluorescence microscopy and showed in merge with bright-field images. One set of representative images of three independent experiments is shown. Scale bars, 50 µm. (D) PI-positive cells in five randomly chosen fields each containing ~100 cells were quantified. The percentage of cell death is defined as the ratio of PI-positive cells relative to all cells (revealed by Hoechst). Data are shown as mean ± SD ( n = 5). * P

    Techniques Used: Activation Assay, Activity Assay, Derivative Assay, Incubation, Western Blot, Expressing, Staining, Fluorescence, Microscopy

    Baicalin attenuated nigericin-induced NOD-like receptor (NLR) family, pyrin containing domain 3 (NLRP3) inflammasome activation. Bone marrow-derived macrophages were first primed with LPS (500 ng/ml) for 4 h and then pretreated with indicated doses of baicalin for 1 h, followed by incubation with nigericin (10 μM) for 1 h without LPS. (A) Western blot analysis was used to assess the expression and secretion of indicated proteins in the cell lysates and culture supernatants, respectively. β-Tubulin was used as a loading control for cell lysates. (B,C) Histograms showing the relative intensity of capase-1p10 (B) or interleukin (IL)-1β (C) bands in culture supernatants in panel (A) . The intensity of capase-1p10 or IL-1β bands in nigericin group was set to 1.0. The intensity of the other groups was calculated relative to the nigericin group. (D) The levels of soluble IL-1β were detected by cytometric bead array assay in the culture supernatants. The experiments were performed three times independently, with one representative experiment shown. Data are shown as mean ± SD ( n = 3). ***P
    Figure Legend Snippet: Baicalin attenuated nigericin-induced NOD-like receptor (NLR) family, pyrin containing domain 3 (NLRP3) inflammasome activation. Bone marrow-derived macrophages were first primed with LPS (500 ng/ml) for 4 h and then pretreated with indicated doses of baicalin for 1 h, followed by incubation with nigericin (10 μM) for 1 h without LPS. (A) Western blot analysis was used to assess the expression and secretion of indicated proteins in the cell lysates and culture supernatants, respectively. β-Tubulin was used as a loading control for cell lysates. (B,C) Histograms showing the relative intensity of capase-1p10 (B) or interleukin (IL)-1β (C) bands in culture supernatants in panel (A) . The intensity of capase-1p10 or IL-1β bands in nigericin group was set to 1.0. The intensity of the other groups was calculated relative to the nigericin group. (D) The levels of soluble IL-1β were detected by cytometric bead array assay in the culture supernatants. The experiments were performed three times independently, with one representative experiment shown. Data are shown as mean ± SD ( n = 3). ***P

    Techniques Used: Activation Assay, Derivative Assay, Incubation, Western Blot, Expressing

    Baicalin suppressed adenosine triphosphate (ATP)-induced activation of NOD-like receptor (NLR) family, pyrin containing domain 3 (NLRP3) inflammasome. Bone marrow-derived macrophages were first primed with LPS (500 ng/ml) for 4 h and then pretreated with graded concentrations of baicalin for 1 h, followed by incubation with ATP (3 mM) for 30 min in the absence of LPS. (A) Western blotting was used to assess the expression levels of indicated proteins in the cell lysates and culture supernatants, respectively. β-Tubulin was used as a loading control for cell lysates. (B,C) Histograms showing the relative intensity of capase-1p10 (B) or interleukin (IL)-1β (C) bands in culture supernatants in (A) . The intensity of capase-1p10 or IL-1β bands in ATP group was set to 1.0. The intensity of the other groups was calculated relative to the ATP group. (D) . Cells were treated as in panel (A) . The levels of soluble IL-1β were detected by cytometric bead array assay in the culture supernatants. The experiments were performed three times independently, with one representative experiment shown. Data are shown as mean ± SD ( n = 3). * P
    Figure Legend Snippet: Baicalin suppressed adenosine triphosphate (ATP)-induced activation of NOD-like receptor (NLR) family, pyrin containing domain 3 (NLRP3) inflammasome. Bone marrow-derived macrophages were first primed with LPS (500 ng/ml) for 4 h and then pretreated with graded concentrations of baicalin for 1 h, followed by incubation with ATP (3 mM) for 30 min in the absence of LPS. (A) Western blotting was used to assess the expression levels of indicated proteins in the cell lysates and culture supernatants, respectively. β-Tubulin was used as a loading control for cell lysates. (B,C) Histograms showing the relative intensity of capase-1p10 (B) or interleukin (IL)-1β (C) bands in culture supernatants in (A) . The intensity of capase-1p10 or IL-1β bands in ATP group was set to 1.0. The intensity of the other groups was calculated relative to the ATP group. (D) . Cells were treated as in panel (A) . The levels of soluble IL-1β were detected by cytometric bead array assay in the culture supernatants. The experiments were performed three times independently, with one representative experiment shown. Data are shown as mean ± SD ( n = 3). * P

    Techniques Used: Activation Assay, Derivative Assay, Incubation, Western Blot, Expressing

    Baicalin administration prolonged mouse survival in bacterial sepsis. (A) C57BL/6 mice were administered (intragastrically) with baicalin (100 or 200 mg/kg body weight) or vehicle (2% Tween-80 in PBS) once 3 h before peritoneal injection with viable Escherichia coli (2 × 10 9 CFU/mouse). One hour after the bacterial injection, mice were intragastrically administered with baicalin or vehicle once again. Mouse survival was monitored every 6 h for five consecutive days. Kaplan–Meier survival curves were used to analyze the data (10 mice per group). The significance was evaluated by the log-rank (Mantel–Cox) test. (B) Mice were treated as in panel (A) . The serum levels of interleukin (IL)-1β at 4 and 8 h post bacterial infection were measured by cytometric bead array assay (five mice per group). ** P
    Figure Legend Snippet: Baicalin administration prolonged mouse survival in bacterial sepsis. (A) C57BL/6 mice were administered (intragastrically) with baicalin (100 or 200 mg/kg body weight) or vehicle (2% Tween-80 in PBS) once 3 h before peritoneal injection with viable Escherichia coli (2 × 10 9 CFU/mouse). One hour after the bacterial injection, mice were intragastrically administered with baicalin or vehicle once again. Mouse survival was monitored every 6 h for five consecutive days. Kaplan–Meier survival curves were used to analyze the data (10 mice per group). The significance was evaluated by the log-rank (Mantel–Cox) test. (B) Mice were treated as in panel (A) . The serum levels of interleukin (IL)-1β at 4 and 8 h post bacterial infection were measured by cytometric bead array assay (five mice per group). ** P

    Techniques Used: Mouse Assay, Injection, Infection

    3) Product Images from "NLRP3 signaling drives macrophage-induced adaptive immune suppression in pancreatic carcinoma"

    Article Title: NLRP3 signaling drives macrophage-induced adaptive immune suppression in pancreatic carcinoma

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20161707

    NLRP3 expression in human and mouse PDA. (A) Lysate from 3-mo-old WT, KC, and KC;NLRP3 −/− mice were tested for expression of IL-1β and IL-18 by Western blotting. Ponceau staining is shown. Experiments were repeated three times. Representative data are shown. (B) Frozen sections of pancreata of mouse PDA tumors were tested for coexpression of CD11b and NLRP3 or CK19 and NLRP3 compared with respective isotype controls. Bar, 10 µm. (C) F4/80 + Gr1 − CD11c − CD11b + macrophages from pancreata or spleen macrophages from 3-mo-old KC mice were tested for expression of NLRP3 compared with isotype controls. (D) Macrophages from pancreata or spleen of KC mice were tested for coexpression of MHC II and CD206. (E) MHC II − CD206 + and MHC II + CD206 − pancreatic macrophage subsets from 3-mo-old KC mice were gated and tested for expression of NLRP3 and IL-1β. Representative and quantitative data are shown. Positive gates are based on isotype controls (not depicted). (F) MHC II − CD206 + and MHC II + CD206 − TAM subsets from WT mice bearing orthotopic PDA were gated and tested for expression of NLRP3 and IL-1β. (G) Macrophages from WT control pancreata or pancreata or spleen of WT mice harboring orthotopic KPC tumors were tested for expression of NLRP3. (H) Paraffin-embedded sections of human PDA were tested for expression of NLRP3 compared with isotype control. Bar, 20 µm. (I) CD15 + monocytic cells from single-cell suspensions of human PDA or PBMCs were gated by flow cytometry and tested for expression of NLRP3. Representative contour plots and quantitative data from six patients are shown. (J) Splenic macrophages from WT mice were cultured alone or in a 5:1 ratio with KPC-derived tumor cells. At 24 h, macrophages were tested for expression of CD206, IL-10, and NLRP3. (K) Similarly, BMDMs from WT mice were stimulated with recombinant TGF-β or TNF and tested for NLRP3 expression. (L) Orthotopic PDA-bearing mice were serially treated with a neutralizing TGF-β mAb or isotype control. Tumors were harvested on day 21, and expression of NLRP3 and CD206 in TAMs was determined by flow cytometry. n = 5/group. All mouse experiments were repeated a minimum of twice using five mice per experimental group. Littermate controls were used. Unpaired Student’s t test was used for statistical analyses. *, P
    Figure Legend Snippet: NLRP3 expression in human and mouse PDA. (A) Lysate from 3-mo-old WT, KC, and KC;NLRP3 −/− mice were tested for expression of IL-1β and IL-18 by Western blotting. Ponceau staining is shown. Experiments were repeated three times. Representative data are shown. (B) Frozen sections of pancreata of mouse PDA tumors were tested for coexpression of CD11b and NLRP3 or CK19 and NLRP3 compared with respective isotype controls. Bar, 10 µm. (C) F4/80 + Gr1 − CD11c − CD11b + macrophages from pancreata or spleen macrophages from 3-mo-old KC mice were tested for expression of NLRP3 compared with isotype controls. (D) Macrophages from pancreata or spleen of KC mice were tested for coexpression of MHC II and CD206. (E) MHC II − CD206 + and MHC II + CD206 − pancreatic macrophage subsets from 3-mo-old KC mice were gated and tested for expression of NLRP3 and IL-1β. Representative and quantitative data are shown. Positive gates are based on isotype controls (not depicted). (F) MHC II − CD206 + and MHC II + CD206 − TAM subsets from WT mice bearing orthotopic PDA were gated and tested for expression of NLRP3 and IL-1β. (G) Macrophages from WT control pancreata or pancreata or spleen of WT mice harboring orthotopic KPC tumors were tested for expression of NLRP3. (H) Paraffin-embedded sections of human PDA were tested for expression of NLRP3 compared with isotype control. Bar, 20 µm. (I) CD15 + monocytic cells from single-cell suspensions of human PDA or PBMCs were gated by flow cytometry and tested for expression of NLRP3. Representative contour plots and quantitative data from six patients are shown. (J) Splenic macrophages from WT mice were cultured alone or in a 5:1 ratio with KPC-derived tumor cells. At 24 h, macrophages were tested for expression of CD206, IL-10, and NLRP3. (K) Similarly, BMDMs from WT mice were stimulated with recombinant TGF-β or TNF and tested for NLRP3 expression. (L) Orthotopic PDA-bearing mice were serially treated with a neutralizing TGF-β mAb or isotype control. Tumors were harvested on day 21, and expression of NLRP3 and CD206 in TAMs was determined by flow cytometry. n = 5/group. All mouse experiments were repeated a minimum of twice using five mice per experimental group. Littermate controls were used. Unpaired Student’s t test was used for statistical analyses. *, P

    Techniques Used: Expressing, Mouse Assay, Western Blot, Staining, Flow Cytometry, Cytometry, Cell Culture, Derivative Assay, Recombinant

    4) Product Images from "Beraprost sodium preconditioning prevents inflammation, apoptosis, and autophagy during hepatic ischemia-reperfusion injury in mice via the P38 and JNK pathways"

    Article Title: Beraprost sodium preconditioning prevents inflammation, apoptosis, and autophagy during hepatic ischemia-reperfusion injury in mice via the P38 and JNK pathways

    Journal: Drug Design, Development and Therapy

    doi: 10.2147/DDDT.S182292

    Probable mechanisms of BPS preconditioning against hepatic IR injury. Notes: In hepatic IR injury, activated KCs released inflammatory cytokines such as TNF-α and IL-1β. TNF-α and IL-1β subsequently activated P38 and JNK phosphorylation, which not only further aggravated inflammation but also promoted intrinsic apoptosis and autophagy. Besides, TNF-α could induce extrinsic apoptosis and intensify intrinsic apoptosis as well. BPS preconditioning afforded protective effects against hepatic IR injury partially via suppressing P38 and JNK phosphorylation to alleviate inflammation, apoptosis and autophagy. Abbreviations: BPS, beraprost sodium; IL-1β, interleukin-1β; IR, ischemia-reperfusion; JNK, c-Jun N-terminal kinase; KCs, Kupffer cells; TNF-α, tumor necrosis factor-α.
    Figure Legend Snippet: Probable mechanisms of BPS preconditioning against hepatic IR injury. Notes: In hepatic IR injury, activated KCs released inflammatory cytokines such as TNF-α and IL-1β. TNF-α and IL-1β subsequently activated P38 and JNK phosphorylation, which not only further aggravated inflammation but also promoted intrinsic apoptosis and autophagy. Besides, TNF-α could induce extrinsic apoptosis and intensify intrinsic apoptosis as well. BPS preconditioning afforded protective effects against hepatic IR injury partially via suppressing P38 and JNK phosphorylation to alleviate inflammation, apoptosis and autophagy. Abbreviations: BPS, beraprost sodium; IL-1β, interleukin-1β; IR, ischemia-reperfusion; JNK, c-Jun N-terminal kinase; KCs, Kupffer cells; TNF-α, tumor necrosis factor-α.

    Techniques Used:

    BPS preconditioning inhibited TNF-α and IL-1β expressions. Notes: ( A ) Relative TNF- α and IL- 1β mRNA levels were determined by qRT-PCR. ( B ) Serum TNF-α and IL-1β were detected by ELISA. ( C ) TNF-α and IL-1β protein expressions in liver tissues at 8 hours after reperfusion are shown by immunohistochemical staining. Final evaluations were made using Image-Pro Plus 6.0 software to calculate the IODs of the positive staining area. ( D ) Western blot analysis of TNF-α and IL-1β protein levels. Gray values were determined by Odyssey software. Relative TNF-α and IL-1β levels were measured through relative changes of gray values. Data are presented as mean ± SD (n=6; * P
    Figure Legend Snippet: BPS preconditioning inhibited TNF-α and IL-1β expressions. Notes: ( A ) Relative TNF- α and IL- 1β mRNA levels were determined by qRT-PCR. ( B ) Serum TNF-α and IL-1β were detected by ELISA. ( C ) TNF-α and IL-1β protein expressions in liver tissues at 8 hours after reperfusion are shown by immunohistochemical staining. Final evaluations were made using Image-Pro Plus 6.0 software to calculate the IODs of the positive staining area. ( D ) Western blot analysis of TNF-α and IL-1β protein levels. Gray values were determined by Odyssey software. Relative TNF-α and IL-1β levels were measured through relative changes of gray values. Data are presented as mean ± SD (n=6; * P

    Techniques Used: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Software, Western Blot

    5) Product Images from "Caspase-8 promotes NLRP1/NLRP3 inflammasome activation and IL-1β production in acute glaucoma"

    Article Title: Caspase-8 promotes NLRP1/NLRP3 inflammasome activation and IL-1β production in acute glaucoma

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1402819111

    Diagram illustrating the proposed pathway by which TLR4/caspase-8/inflammasome up-regulation induces the processing of IL-1β and promotes the inflammatory response in acute glaucoma.
    Figure Legend Snippet: Diagram illustrating the proposed pathway by which TLR4/caspase-8/inflammasome up-regulation induces the processing of IL-1β and promotes the inflammatory response in acute glaucoma.

    Techniques Used:

    Caspase-8 signaling was required for processing of IL-1β only partially through caspase-1–dependent NLRP1 and NLRP3 inflammasome activation. Gene expression ( A ) ( n = 10) and protein expression of IL-1β ( B ) ( n = 15) were elevated in ischemic retina at different time points after reperfusion. ( C ) The intravitreous injection of caspase-8 inhibitor, Z-IETD-fmk (20 μM), completely inhibited the processing of IL-1β ( n = 9). ( D ) The intravitreous injection of caspase-1 inhibitor, Z-YVAD-fmk (20 μM), partially reduced the processing of IL-1β ( n = 9). ( E and F ) Inhibition of caspase-1 attenuated the damage and RGC death from retinal ischemia ( n = 12). Representative images are shown. Data are presented as mean ± SD; * P
    Figure Legend Snippet: Caspase-8 signaling was required for processing of IL-1β only partially through caspase-1–dependent NLRP1 and NLRP3 inflammasome activation. Gene expression ( A ) ( n = 10) and protein expression of IL-1β ( B ) ( n = 15) were elevated in ischemic retina at different time points after reperfusion. ( C ) The intravitreous injection of caspase-8 inhibitor, Z-IETD-fmk (20 μM), completely inhibited the processing of IL-1β ( n = 9). ( D ) The intravitreous injection of caspase-1 inhibitor, Z-YVAD-fmk (20 μM), partially reduced the processing of IL-1β ( n = 9). ( E and F ) Inhibition of caspase-1 attenuated the damage and RGC death from retinal ischemia ( n = 12). Representative images are shown. Data are presented as mean ± SD; * P

    Techniques Used: Activation Assay, Expressing, Injection, Inhibition

    6) Product Images from "IL-2/Anti-IL-2 Complex Attenuates Inflammation and BBB Disruption in Mice Subjected to Traumatic Brain Injury"

    Article Title: IL-2/Anti-IL-2 Complex Attenuates Inflammation and BBB Disruption in Mice Subjected to Traumatic Brain Injury

    Journal: Frontiers in Neurology

    doi: 10.3389/fneur.2017.00281

    Post-injury IL-2C administration suppressed the expression of M1-associated markers around the lesion area. (A) Co-localization of CD16/32 with the microglia marker Iba1 in the contusion area on day 3 post-injury showed that treatment with IL-2C markedly reduced the amount of M1 microglia. (B) Cell count analyses indicated that IL-2C significantly reduced the number of CD16/32 and Iba-1 double-positive cells. (C–E) The traumatic brain injury (TBI)-induced expression of IL-1β and TNF-α in the ipsilateral cortices on days 1 and 3 post-injury was inhibited by treatment with IL-2C. Data are presented as the mean ± SD; * p
    Figure Legend Snippet: Post-injury IL-2C administration suppressed the expression of M1-associated markers around the lesion area. (A) Co-localization of CD16/32 with the microglia marker Iba1 in the contusion area on day 3 post-injury showed that treatment with IL-2C markedly reduced the amount of M1 microglia. (B) Cell count analyses indicated that IL-2C significantly reduced the number of CD16/32 and Iba-1 double-positive cells. (C–E) The traumatic brain injury (TBI)-induced expression of IL-1β and TNF-α in the ipsilateral cortices on days 1 and 3 post-injury was inhibited by treatment with IL-2C. Data are presented as the mean ± SD; * p

    Techniques Used: Expressing, Marker, Cell Counting

    7) Product Images from "Au Clusters Treat Rheumatoid Arthritis with Uniquely Reversing Cartilage/Bone Destruction"

    Article Title: Au Clusters Treat Rheumatoid Arthritis with Uniquely Reversing Cartilage/Bone Destruction

    Journal: Advanced Science

    doi: 10.1002/advs.201801671

    Au clusters synthesized with glutathione as the template (Au 29 SG 27 , designated GA) inhibit the inflammatory response of lipopolysaccharide (LPS )‐stimulated RAW 264.7 cells. a–e) Concentration‐dependent inhibition of inflammatory mediators NO a), TNF‐α b), IL‐1β c), IL‐6 d), and PGE 2 e) in LPS‐stimulated RAW 264.7 cells treated with different concentrations of Au clusters. NO released in the cell supernatants was measured using Griess reagent and an ELISA plate reader. TNF‐α, IL‐1β, IL‐6, and PGE 2 were measured by ELISA analysis. Data ( n = 6, mean ± SD) are representative of at least two independent experiments. f) Concentration‐dependent inhibition of the i‐NOS, COX‐2, TNF‐α, IL‐1β, and IL‐6 protein levels in LPS‐stimulated RAW 264.7 cells measured by western blot analysis. β‐actin was used as an internal control. Images are representative of two experiments. g) Concentration‐dependent inhibition of the i‐NOS, COX‐2, TNF‐α, IL‐1β, and IL‐6 mRNA levels in LPS‐stimulated RAW 264.7 cells analyzed by real‐time RT‐PCR using specific primers. β‐actin was used as an internal control. Images are representative of two experiments. h) Inhibitory effect of different concentration of Au clusters on IKK, IκBα, and p65 phosphorylation in LPS‐stimulated RAW 264.7 cells analyzed by western blotting. β‐actin was used as an internal control. Images are representative of two experiments. i) Inhibitory effect of different concentrations of Au clusters on MAPK signaling pathways in LPS‐stimulated RAW 264.7 cells analyzed by western blotting. β‐actin was used as an internal control. Images are representative of two experiments. Data are presented as the mean ± SD; * p
    Figure Legend Snippet: Au clusters synthesized with glutathione as the template (Au 29 SG 27 , designated GA) inhibit the inflammatory response of lipopolysaccharide (LPS )‐stimulated RAW 264.7 cells. a–e) Concentration‐dependent inhibition of inflammatory mediators NO a), TNF‐α b), IL‐1β c), IL‐6 d), and PGE 2 e) in LPS‐stimulated RAW 264.7 cells treated with different concentrations of Au clusters. NO released in the cell supernatants was measured using Griess reagent and an ELISA plate reader. TNF‐α, IL‐1β, IL‐6, and PGE 2 were measured by ELISA analysis. Data ( n = 6, mean ± SD) are representative of at least two independent experiments. f) Concentration‐dependent inhibition of the i‐NOS, COX‐2, TNF‐α, IL‐1β, and IL‐6 protein levels in LPS‐stimulated RAW 264.7 cells measured by western blot analysis. β‐actin was used as an internal control. Images are representative of two experiments. g) Concentration‐dependent inhibition of the i‐NOS, COX‐2, TNF‐α, IL‐1β, and IL‐6 mRNA levels in LPS‐stimulated RAW 264.7 cells analyzed by real‐time RT‐PCR using specific primers. β‐actin was used as an internal control. Images are representative of two experiments. h) Inhibitory effect of different concentration of Au clusters on IKK, IκBα, and p65 phosphorylation in LPS‐stimulated RAW 264.7 cells analyzed by western blotting. β‐actin was used as an internal control. Images are representative of two experiments. i) Inhibitory effect of different concentrations of Au clusters on MAPK signaling pathways in LPS‐stimulated RAW 264.7 cells analyzed by western blotting. β‐actin was used as an internal control. Images are representative of two experiments. Data are presented as the mean ± SD; * p

    Techniques Used: Synthesized, Concentration Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR

    Au clusters inhibit inflammatory responses in rat CIA. a–c) Progression of ankle circumference a), clinical arthritis score b), and body weight c) in CIA rats ( n = 10) over 6 weeks after starting treatment with vehicle (buffer), MTX, and Au clusters. Nonimmunized normal rats treated with vehicle were used as a control group. d) Representative photographs of CIA rats treated with vehicle, MTX, and Au clusters and nonimmunized normal rats at the 1) initial, 2) middle, and 3) end timepoints of drug administration. e,f) The levels of proinflammatory cytokines (TNF‐a, IL‐1β, and IL‐6) and PGE 2 at the end of the study (day 42) in the serum e) and articular tissue f) of CIA rats treated with vehicle (buffer), MTX, and Au clusters. The cytokine and PGE 2 levels of nonimmunized rats in serum and articular tissue were used as controls. Data are presented as the mean ± SD; # p
    Figure Legend Snippet: Au clusters inhibit inflammatory responses in rat CIA. a–c) Progression of ankle circumference a), clinical arthritis score b), and body weight c) in CIA rats ( n = 10) over 6 weeks after starting treatment with vehicle (buffer), MTX, and Au clusters. Nonimmunized normal rats treated with vehicle were used as a control group. d) Representative photographs of CIA rats treated with vehicle, MTX, and Au clusters and nonimmunized normal rats at the 1) initial, 2) middle, and 3) end timepoints of drug administration. e,f) The levels of proinflammatory cytokines (TNF‐a, IL‐1β, and IL‐6) and PGE 2 at the end of the study (day 42) in the serum e) and articular tissue f) of CIA rats treated with vehicle (buffer), MTX, and Au clusters. The cytokine and PGE 2 levels of nonimmunized rats in serum and articular tissue were used as controls. Data are presented as the mean ± SD; # p

    Techniques Used:

    8) Product Images from "Nrf2 signaling and autophagy are complementary in protecting lipopolysaccharide/d-galactosamine-induced acute liver injury by licochalcone A"

    Article Title: Nrf2 signaling and autophagy are complementary in protecting lipopolysaccharide/d-galactosamine-induced acute liver injury by licochalcone A

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-1543-z

    Effects of Lico A-treated on LPS/GalN-induced Txnip-NLRP3 inflammasome signaling pathway in ALI mice. Liver tissues were collected from the mice 3 h and 6 h after LPS/GalN challenge and analyzed by western blot. a Effects of Lico A treatment on LPS/GalN-induced Txinp, Trx-1, NLRP3, ASC, Cleaved-caspase-1, and Mature-IL-1β were measured by western blotting. b – g Quantification of relative protein expression was performed by densitometric analysis and β-actin was used as an internal control. Similar results were obtained from three independent experiments. All data are presented as means±SEM ( n = 5/group). * p
    Figure Legend Snippet: Effects of Lico A-treated on LPS/GalN-induced Txnip-NLRP3 inflammasome signaling pathway in ALI mice. Liver tissues were collected from the mice 3 h and 6 h after LPS/GalN challenge and analyzed by western blot. a Effects of Lico A treatment on LPS/GalN-induced Txinp, Trx-1, NLRP3, ASC, Cleaved-caspase-1, and Mature-IL-1β were measured by western blotting. b – g Quantification of relative protein expression was performed by densitometric analysis and β-actin was used as an internal control. Similar results were obtained from three independent experiments. All data are presented as means±SEM ( n = 5/group). * p

    Techniques Used: Mouse Assay, Western Blot, Expressing

    Effect of Lico A-treated on the secretion of TNF-α, IL-6, and IL-1β in mice with LPS/GalN-induced ALI. Mice at 3 h or 6 h after LPS/GalN injection, serum of mice were collected for assessment of production of inflammatory cytokines. a – c Effects of Lico A on LPS/GalN-induced serum TNF-α, IL-6, and IL-1β generation. Similar results were obtained from three independent experiments. All data are presented as means±SEM ( n = 5/group). ** p
    Figure Legend Snippet: Effect of Lico A-treated on the secretion of TNF-α, IL-6, and IL-1β in mice with LPS/GalN-induced ALI. Mice at 3 h or 6 h after LPS/GalN injection, serum of mice were collected for assessment of production of inflammatory cytokines. a – c Effects of Lico A on LPS/GalN-induced serum TNF-α, IL-6, and IL-1β generation. Similar results were obtained from three independent experiments. All data are presented as means±SEM ( n = 5/group). ** p

    Techniques Used: Mouse Assay, Injection

    9) Product Images from "Baicalein ameliorates TNBS-induced colitis by suppressing TLR4/MyD88 signaling cascade and NLRP3 inflammasome activation in mice"

    Article Title: Baicalein ameliorates TNBS-induced colitis by suppressing TLR4/MyD88 signaling cascade and NLRP3 inflammasome activation in mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-12562-6

    Baicalein inhibited NF-κB pathway in vitro . Cells were treated with dose range of baicalein for 2 h prior to LPS (1 µg/ml) treatment for an additional 24 h. ( A ) The production of NO in RAW264.7 cells induced by LPS was measured as described in the Methods. ( B ) Protein level of RAW264.7 cells was determined with antibody against iNOS (1:1000) and β-actin (1:2000 dilution) by immunoblotting. Quantification of the protein expression was performed by densitometric analysis of the blots. Expression was normalized to β-actin. ( C ) NF-κB promoter-driven luciferase activity in RAW264.7 cells was determined using a luciferase assay system as described in the Methods. Results were expressed as fold values of control cells. ( D ) mRNA expression of iNOS, COX-2, IL-1α and IL-1β in THP-1 cells was determined by qRT-PCR. Expression was normalized to β-actin. ( E ) NF-κB p65 nuclear translocation in RAW264.7 cells was evaluated by immunofluorescence staining and images were captured by a fluorescence microscope. Scale bar corresponds to 50 μm and applies throughout. Data were expressed as mean ± SD of three independent experiments (n = 3). ### p
    Figure Legend Snippet: Baicalein inhibited NF-κB pathway in vitro . Cells were treated with dose range of baicalein for 2 h prior to LPS (1 µg/ml) treatment for an additional 24 h. ( A ) The production of NO in RAW264.7 cells induced by LPS was measured as described in the Methods. ( B ) Protein level of RAW264.7 cells was determined with antibody against iNOS (1:1000) and β-actin (1:2000 dilution) by immunoblotting. Quantification of the protein expression was performed by densitometric analysis of the blots. Expression was normalized to β-actin. ( C ) NF-κB promoter-driven luciferase activity in RAW264.7 cells was determined using a luciferase assay system as described in the Methods. Results were expressed as fold values of control cells. ( D ) mRNA expression of iNOS, COX-2, IL-1α and IL-1β in THP-1 cells was determined by qRT-PCR. Expression was normalized to β-actin. ( E ) NF-κB p65 nuclear translocation in RAW264.7 cells was evaluated by immunofluorescence staining and images were captured by a fluorescence microscope. Scale bar corresponds to 50 μm and applies throughout. Data were expressed as mean ± SD of three independent experiments (n = 3). ### p

    Techniques Used: In Vitro, Expressing, Luciferase, Activity Assay, Quantitative RT-PCR, Translocation Assay, Immunofluorescence, Staining, Fluorescence, Microscopy

    Baicalein inhibited NF-κB pathway in TNBS-induced mice. ( A ) Colon segments from mice were excised and homogenized. The supernatants were assayed for the determination of the activity of MPO. ( B ) mRNA expression of iNOS, ICAM-1, MCP-1, COX-2, TNF-α, and IL-1β in colonic tissue was determined by qRT-PCR. Expression was normalized to β-actin. ( C ) Representative images of p-p65 immunostaining in colonic tissue. Scale bar corresponds to 50 μm and applies throughout. ( D ) The mean intensity of p-p65 staining was determined by image analysis and was represented as optical density. Data were expressed as mean ± SD (n = 6). ### p
    Figure Legend Snippet: Baicalein inhibited NF-κB pathway in TNBS-induced mice. ( A ) Colon segments from mice were excised and homogenized. The supernatants were assayed for the determination of the activity of MPO. ( B ) mRNA expression of iNOS, ICAM-1, MCP-1, COX-2, TNF-α, and IL-1β in colonic tissue was determined by qRT-PCR. Expression was normalized to β-actin. ( C ) Representative images of p-p65 immunostaining in colonic tissue. Scale bar corresponds to 50 μm and applies throughout. ( D ) The mean intensity of p-p65 staining was determined by image analysis and was represented as optical density. Data were expressed as mean ± SD (n = 6). ### p

    Techniques Used: Mouse Assay, Activity Assay, Expressing, Quantitative RT-PCR, Immunostaining, Staining

    Baicalein inhibited MAPK signaling molecules in RAW264.7 cells and inhibited NLRP3 inflammasome activation in THP-1 cells. Cells were treated with baicalein for 2 h followed by an additional treatment with or without LPS (1 μg/ml) for 24 h. ( A ) Protein levels in RAW264.7 cells were determined with antibodies against JNK, p-JNK, ERK1/2, p-ERK1/2, p38, p-p38 (1:1000 dilution) and β-actin (1:2000 dilution) by immunoblotting. Quantification of the protein expression was performed by densitometric analysis of the blots. The ratio of phosphorylated MAPK to regular MAPK was shown ( B ). ( C ) Protein levels in THP-1 cells were determined with antibodies against NLRP3, ASC, caspase-1 (1:1000 dilution) and β-actin (1:2000 dilution) by immunoblotting. Quantification of the protein expression was performed by densitometric analysis of the blots ( D ). ( E ) THP-1 cells were pretreated with baicalein for 2 h and then followed by stimulation with ATP (5 mM) for 24 h. Protein expression was determined with antibodies against NLRP3, IL-1β (1:1000 dilution) and β-actin (1:2000 dilution) by immunoblotting. Quantification of the protein expression was performed by densitometric analysis of the blots ( F ). Expression was normalized to β-actin. Results were expressed as means ± SD of three independent experiments (n = 3). # p
    Figure Legend Snippet: Baicalein inhibited MAPK signaling molecules in RAW264.7 cells and inhibited NLRP3 inflammasome activation in THP-1 cells. Cells were treated with baicalein for 2 h followed by an additional treatment with or without LPS (1 μg/ml) for 24 h. ( A ) Protein levels in RAW264.7 cells were determined with antibodies against JNK, p-JNK, ERK1/2, p-ERK1/2, p38, p-p38 (1:1000 dilution) and β-actin (1:2000 dilution) by immunoblotting. Quantification of the protein expression was performed by densitometric analysis of the blots. The ratio of phosphorylated MAPK to regular MAPK was shown ( B ). ( C ) Protein levels in THP-1 cells were determined with antibodies against NLRP3, ASC, caspase-1 (1:1000 dilution) and β-actin (1:2000 dilution) by immunoblotting. Quantification of the protein expression was performed by densitometric analysis of the blots ( D ). ( E ) THP-1 cells were pretreated with baicalein for 2 h and then followed by stimulation with ATP (5 mM) for 24 h. Protein expression was determined with antibodies against NLRP3, IL-1β (1:1000 dilution) and β-actin (1:2000 dilution) by immunoblotting. Quantification of the protein expression was performed by densitometric analysis of the blots ( F ). Expression was normalized to β-actin. Results were expressed as means ± SD of three independent experiments (n = 3). # p

    Techniques Used: Activation Assay, Expressing

    10) Product Images from "Curcumin Reduces Neuronal Loss and Inhibits the NLRP3 Inflammasome Activation in an Epileptic Rat Model"

    Article Title: Curcumin Reduces Neuronal Loss and Inhibits the NLRP3 Inflammasome Activation in an Epileptic Rat Model

    Journal: Current Neurovascular Research

    doi: 10.2174/1567202615666180731100224

    Curcumin attenuated inflammation and neuronal death via modulating the activation of NLPR3/inflammasome in epilepsy. ( A ), Representative Western blot analysis of IL-1β, NLRP3 and the cleavage of caspase-1 and protein expression in hippocampi from rats with/without curcumin treatment after KA induced epilepsy. ( B ), Quantification of the level of NLRP3 and cleavage of caspase-1 and IL-1β. ( C ), Neurons number in hippocampus was detected with H E staining. The results represent the mean ± SEM for three experiments. P
    Figure Legend Snippet: Curcumin attenuated inflammation and neuronal death via modulating the activation of NLPR3/inflammasome in epilepsy. ( A ), Representative Western blot analysis of IL-1β, NLRP3 and the cleavage of caspase-1 and protein expression in hippocampi from rats with/without curcumin treatment after KA induced epilepsy. ( B ), Quantification of the level of NLRP3 and cleavage of caspase-1 and IL-1β. ( C ), Neurons number in hippocampus was detected with H E staining. The results represent the mean ± SEM for three experiments. P

    Techniques Used: Activation Assay, Western Blot, Expressing, Staining

    Inflammasome was activated in KA induced epilepsy rats. ( A ), Representative Western blot analysis of IL-1β, NLRP3 and the cleavage of caspase-1 and protein expression in hippocampi from control group or animals after and KA induced epilepsy. ( B ), Quantification of the level of NLRP3 and cleavage of caspase-1 and IL-1β. ( C ), NLRP3 and Iba1 expression in hippocampus was detected with immunochemistry.The results represent the mean ± SEM for three experiments. P
    Figure Legend Snippet: Inflammasome was activated in KA induced epilepsy rats. ( A ), Representative Western blot analysis of IL-1β, NLRP3 and the cleavage of caspase-1 and protein expression in hippocampi from control group or animals after and KA induced epilepsy. ( B ), Quantification of the level of NLRP3 and cleavage of caspase-1 and IL-1β. ( C ), NLRP3 and Iba1 expression in hippocampus was detected with immunochemistry.The results represent the mean ± SEM for three experiments. P

    Techniques Used: Western Blot, Expressing

    11) Product Images from "Propofol Promotes Ankle Fracture Healing in Children by Inhibiting Inflammatory Response"

    Article Title: Propofol Promotes Ankle Fracture Healing in Children by Inhibiting Inflammatory Response

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.908592

    The levels of TNF-α, IL-1β, and IL-6 significantly enhanced in children with fractured ankle. The mRNA and protein levels of TNF-α, IL-1β and IL-6 in children with or without fractured ankle were detected by qRT-PCR and ELISA, respectively. Controls: healthy children; Patients: children with fractured ankle. **, p
    Figure Legend Snippet: The levels of TNF-α, IL-1β, and IL-6 significantly enhanced in children with fractured ankle. The mRNA and protein levels of TNF-α, IL-1β and IL-6 in children with or without fractured ankle were detected by qRT-PCR and ELISA, respectively. Controls: healthy children; Patients: children with fractured ankle. **, p

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

    Propofol significantly inhibits BK-induced increase of TNF-α, IL-1β, and IL-6. After treatment with 1, 5, and 10 μg/ml propofol, mRNA and protein levels of TNF-α, IL-1β, and IL-6 were detected by qRT-PCR and Western blot analysis, respectively. Con – control group; BK – 1 μM BK treatment group; BK + P1 – 1 μM BK + 1 μg/ml propofol treatment group; BK + P5 – 1 μM BK + 5 μg/ml propofol treatment group; BK + P10 – 1 μM BK + 10 μg/ml propofol treatment group. ** p
    Figure Legend Snippet: Propofol significantly inhibits BK-induced increase of TNF-α, IL-1β, and IL-6. After treatment with 1, 5, and 10 μg/ml propofol, mRNA and protein levels of TNF-α, IL-1β, and IL-6 were detected by qRT-PCR and Western blot analysis, respectively. Con – control group; BK – 1 μM BK treatment group; BK + P1 – 1 μM BK + 1 μg/ml propofol treatment group; BK + P5 – 1 μM BK + 5 μg/ml propofol treatment group; BK + P10 – 1 μM BK + 10 μg/ml propofol treatment group. ** p

    Techniques Used: Quantitative RT-PCR, Western Blot

    12) Product Images from "Augmenter of liver regeneration (ALR) regulates acute pancreatitis via inhibiting HMGB1/TLR4/NF-κB signaling pathway"

    Article Title: Augmenter of liver regeneration (ALR) regulates acute pancreatitis via inhibiting HMGB1/TLR4/NF-κB signaling pathway

    Journal: American Journal of Translational Research

    doi:

    Release of TNF-α, IL-1β and MCP-1 in pancreas and/or plasma after individual treatments. Enzyme-linked immunosorbent assay (ELISA) data of pancreas of TNF-α (A), IL-β (C) and MCP-1 (E). ELISA-detected plasma levels of TNF-α (B) and IL-β (D). Data are represented as mean ± SD (n = 8 per group). * P
    Figure Legend Snippet: Release of TNF-α, IL-1β and MCP-1 in pancreas and/or plasma after individual treatments. Enzyme-linked immunosorbent assay (ELISA) data of pancreas of TNF-α (A), IL-β (C) and MCP-1 (E). ELISA-detected plasma levels of TNF-α (B) and IL-β (D). Data are represented as mean ± SD (n = 8 per group). * P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    13) Product Images from "TMEM126B deficiency reduces mitochondrial SDH oxidation by LPS, attenuating HIF-1α stabilization and IL-1β expression"

    Article Title: TMEM126B deficiency reduces mitochondrial SDH oxidation by LPS, attenuating HIF-1α stabilization and IL-1β expression

    Journal: Redox Biology

    doi: 10.1016/j.redox.2018.10.007

    HIF-1α induction after SDH inhibition. A. Succinate dehydrogenase (SDH) activity assay in THP-1 cells treated with the SDH inhibitor atpenin A5 (AA5). B. Time-dependent Western analysis of hypoxia inducible factor (HIF)-1α and tubulin in AA5-treated cells. C. Quantification of B (n = 4). D. THP-1 cells incubated for 6 h with dimethyl succinate (DMS) followed by Western analysis of HIF-1α and tubulin. E. Quantification of D (n = 5). F-H. mRNA expression of glucose transporter 1 (Glut1, F), CL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3, G), and interleukin-1β (IL-1β, H) normalized to the TATA Box binding protein (TBP) after AA5-treatment (4 h) (n = 7). I. IL-1β mRNA analysis of AA5-treated control (shC, black bars) and HIF-1α knockdown (sh1, grey bars) cells (n = 4). J. THP-1 cells were incubated with LPS and TEMPO followed by IL-1β mRNA detection (n = 5). K. THP-1 cells were time-dependently treated with LPS followed by HIF-1α mRNA analysis (n = 7). L. Cells were time-dependently treated with LPS, followed by Western analysis of HIF-1α and tubulin. M. Quantification of L (n = 5). Data are mean values ± SEM, *p
    Figure Legend Snippet: HIF-1α induction after SDH inhibition. A. Succinate dehydrogenase (SDH) activity assay in THP-1 cells treated with the SDH inhibitor atpenin A5 (AA5). B. Time-dependent Western analysis of hypoxia inducible factor (HIF)-1α and tubulin in AA5-treated cells. C. Quantification of B (n = 4). D. THP-1 cells incubated for 6 h with dimethyl succinate (DMS) followed by Western analysis of HIF-1α and tubulin. E. Quantification of D (n = 5). F-H. mRNA expression of glucose transporter 1 (Glut1, F), CL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3, G), and interleukin-1β (IL-1β, H) normalized to the TATA Box binding protein (TBP) after AA5-treatment (4 h) (n = 7). I. IL-1β mRNA analysis of AA5-treated control (shC, black bars) and HIF-1α knockdown (sh1, grey bars) cells (n = 4). J. THP-1 cells were incubated with LPS and TEMPO followed by IL-1β mRNA detection (n = 5). K. THP-1 cells were time-dependently treated with LPS followed by HIF-1α mRNA analysis (n = 7). L. Cells were time-dependently treated with LPS, followed by Western analysis of HIF-1α and tubulin. M. Quantification of L (n = 5). Data are mean values ± SEM, *p

    Techniques Used: Inhibition, Activity Assay, Western Blot, Incubation, Expressing, Binding Assay

    IL-β expression in TMEM126B knockdown cells. A. Interleukin-1β (IL-1β) mRNA analysis in control (shC, black bars) and TMEM126B knockdown cells (sh126B, white bars) treated with LPS for indicated times (n = 7). B. shC, HIF-1α knockdown (sh1, grey bars), and sh126B cells were treated with LPS for 8 h, while atpenin A5 (AA5) was added for the last 4 h. IL-1β mRNA was analyzed (n = 7). C. Western analysis of IL-1β and tubulin in shC and sh126B cells treated with LPS for 8 h. AA5 was added for the last 4 h. D. Quantification of C (n = 7). E. Quantification of cytometric bead array for IL-1β in supernatants from experiments described in C (n = 5). F. Scheme of the proposed two-staged mechanism of IL-1β expression. Data are mean values ± SEM, *p
    Figure Legend Snippet: IL-β expression in TMEM126B knockdown cells. A. Interleukin-1β (IL-1β) mRNA analysis in control (shC, black bars) and TMEM126B knockdown cells (sh126B, white bars) treated with LPS for indicated times (n = 7). B. shC, HIF-1α knockdown (sh1, grey bars), and sh126B cells were treated with LPS for 8 h, while atpenin A5 (AA5) was added for the last 4 h. IL-1β mRNA was analyzed (n = 7). C. Western analysis of IL-1β and tubulin in shC and sh126B cells treated with LPS for 8 h. AA5 was added for the last 4 h. D. Quantification of C (n = 7). E. Quantification of cytometric bead array for IL-1β in supernatants from experiments described in C (n = 5). F. Scheme of the proposed two-staged mechanism of IL-1β expression. Data are mean values ± SEM, *p

    Techniques Used: Expressing, Western Blot

    14) Product Images from "Differences in signaling pathways by IL-1? and IL-18"

    Article Title: Differences in signaling pathways by IL-1? and IL-18

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0402800101

    Effect of IL-1β or IL-18 on NF-κB and AP-1-signaling pathways. A549-Rβ cells were transiently cotransfected with NF-κB( A ) or AP-1 ( B ) luciferase reporter plasmids or control pRL-TK Renilla vector at a ratio of 50:1 and cultured for 24 h. After stimulation with the indicated concentrations of IL-1β or IL-18 for 24 h, supernatants were collected for IL-8 assay ( A Inset ), and cells were harvested for luciferase assay. The data obtained were evaluated as firefly luciferase activity/Renilla luciferase activity. The data represent the mean ± SEM, n = 6. * , P
    Figure Legend Snippet: Effect of IL-1β or IL-18 on NF-κB and AP-1-signaling pathways. A549-Rβ cells were transiently cotransfected with NF-κB( A ) or AP-1 ( B ) luciferase reporter plasmids or control pRL-TK Renilla vector at a ratio of 50:1 and cultured for 24 h. After stimulation with the indicated concentrations of IL-1β or IL-18 for 24 h, supernatants were collected for IL-8 assay ( A Inset ), and cells were harvested for luciferase assay. The data obtained were evaluated as firefly luciferase activity/Renilla luciferase activity. The data represent the mean ± SEM, n = 6. * , P

    Techniques Used: Luciferase, Plasmid Preparation, Cell Culture, Activity Assay

    Effect of IL-18 on IL-8 and PGE 2 production in primary human cells. Human PBMC from three donors were incubated with IL-1β (10 ng/ml) or IL-18 (50 ng/ml) in the absence or presence of IL-1Ra (10 μg/ml). After 24 h, IL-8 was measured in the supernatants, and PGE 2 was measured after 48 h. Mean levels (± SEM) are shown.
    Figure Legend Snippet: Effect of IL-18 on IL-8 and PGE 2 production in primary human cells. Human PBMC from three donors were incubated with IL-1β (10 ng/ml) or IL-18 (50 ng/ml) in the absence or presence of IL-1Ra (10 μg/ml). After 24 h, IL-8 was measured in the supernatants, and PGE 2 was measured after 48 h. Mean levels (± SEM) are shown.

    Techniques Used: Incubation

    IκB degradation after IL-1β or IL-18 stimulation. ( A ) Western blot analysis of IκB was performed with cell extracts from A549-Rβ, immediately following stimulation with IL-1β (10 ng/ml) or IL-18 (50 ng/ml) for indicated times. The amount of protein loading was determined by probing the same filter with antiactin Ab. ( B ) A549-Rβ cells were preincubated for 1 h with increasing concentrations of SN-50 and then stimulated with either IL-1β (10 ng/ml) or IL-18 (50 ng/ml). After 24 h, the supernatants were collected for IL-8 assay. The data represent the mean ± SEM, n = 3.
    Figure Legend Snippet: IκB degradation after IL-1β or IL-18 stimulation. ( A ) Western blot analysis of IκB was performed with cell extracts from A549-Rβ, immediately following stimulation with IL-1β (10 ng/ml) or IL-18 (50 ng/ml) for indicated times. The amount of protein loading was determined by probing the same filter with antiactin Ab. ( B ) A549-Rβ cells were preincubated for 1 h with increasing concentrations of SN-50 and then stimulated with either IL-1β (10 ng/ml) or IL-18 (50 ng/ml). After 24 h, the supernatants were collected for IL-8 assay. The data represent the mean ± SEM, n = 3.

    Techniques Used: Western Blot

    Steady-state COX-2 and IL-8 mRNA levels. A549-Rβ were stimulated with IL-1β or IL-18 in the absence or presence of IL-1Ra (10 μg/ml) added 1 h before cytokine stimulation. Total RNA was extracted and analyzed by RT-PCR for COX-2 and IL-8 after 4 h ( Upper ) or 24 h ( Lower ). The data are from a single experiment representative of four similar studies.
    Figure Legend Snippet: Steady-state COX-2 and IL-8 mRNA levels. A549-Rβ were stimulated with IL-1β or IL-18 in the absence or presence of IL-1Ra (10 μg/ml) added 1 h before cytokine stimulation. Total RNA was extracted and analyzed by RT-PCR for COX-2 and IL-8 after 4 h ( Upper ) or 24 h ( Lower ). The data are from a single experiment representative of four similar studies.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    COX-2 and PGE 2 production in IL-1β- or IL-18-stimulated cells. ( A ) A549-Rβ cells were stimulated with IL-1β (10 ng/ml) or IL-18 (50 ng/ml), and Western blot analysis was performed after 12 ( A ) or 30 ( B ) h. ( B ) PGE 2 levels in supernatants of cells stimulated with the same concentrations of cytokines for 48 h in the absence or presence of IL-1Ra (10 μg/ml). The data represent the mean ± SEM, n = 3. * , P
    Figure Legend Snippet: COX-2 and PGE 2 production in IL-1β- or IL-18-stimulated cells. ( A ) A549-Rβ cells were stimulated with IL-1β (10 ng/ml) or IL-18 (50 ng/ml), and Western blot analysis was performed after 12 ( A ) or 30 ( B ) h. ( B ) PGE 2 levels in supernatants of cells stimulated with the same concentrations of cytokines for 48 h in the absence or presence of IL-1Ra (10 μg/ml). The data represent the mean ± SEM, n = 3. * , P

    Techniques Used: Western Blot

    Phosphorylation and inhibition of p38 MAPK. ( A and B ) Western blot analysis by antiphospho-p38 Ab. Cell extracts were prepared after stimulation with IL-1β (10 ng/ml) and IL-18 (50 ng/ml) for indicated times by trypsinization ( A ). Direct lysis in Triton buffer was used to prepare cell lysates ( B ). ( C ) A549-Rβ cells were preincubated for 1 h with increasing concentrations (0.5–2 μM) of the p38 inhibitor, SB203580, and stimulated with IL-1β or IL-18 at the same concentration in A and B . After 24 h, the supernatants were removed and assayed for IL-8 concentration by enhanced chemiluminescence. Data represent the mean ± SEM ( n = 3).
    Figure Legend Snippet: Phosphorylation and inhibition of p38 MAPK. ( A and B ) Western blot analysis by antiphospho-p38 Ab. Cell extracts were prepared after stimulation with IL-1β (10 ng/ml) and IL-18 (50 ng/ml) for indicated times by trypsinization ( A ). Direct lysis in Triton buffer was used to prepare cell lysates ( B ). ( C ) A549-Rβ cells were preincubated for 1 h with increasing concentrations (0.5–2 μM) of the p38 inhibitor, SB203580, and stimulated with IL-1β or IL-18 at the same concentration in A and B . After 24 h, the supernatants were removed and assayed for IL-8 concentration by enhanced chemiluminescence. Data represent the mean ± SEM ( n = 3).

    Techniques Used: Inhibition, Western Blot, Lysis, Concentration Assay

    IL-18-induced cytokines is independent of IL-1. A549-Rβ cells were stimulated with either IL-1β (10 ng/ml) or IL-18 (50 ng/ml) in the absence or presence of IL-1Ra (10 μg/ml), and after 24 h, the supernatants were assayed for IL-6 ( B ) and IL-8 ( C ). The cells were lysed and assayed for intracellular IL-1α ( A ). The data are the mean ± SEM of nine experiments. * , P
    Figure Legend Snippet: IL-18-induced cytokines is independent of IL-1. A549-Rβ cells were stimulated with either IL-1β (10 ng/ml) or IL-18 (50 ng/ml) in the absence or presence of IL-1Ra (10 μg/ml), and after 24 h, the supernatants were assayed for IL-6 ( B ) and IL-8 ( C ). The cells were lysed and assayed for intracellular IL-1α ( A ). The data are the mean ± SEM of nine experiments. * , P

    Techniques Used:

    15) Product Images from "Magnesium ion influx reduces neuroinflammation in Aβ precursor protein/Presenilin 1 transgenic mice by suppressing the expression of interleukin-1β"

    Article Title: Magnesium ion influx reduces neuroinflammation in Aβ precursor protein/Presenilin 1 transgenic mice by suppressing the expression of interleukin-1β

    Journal: Cellular and Molecular Immunology

    doi: 10.1038/cmi.2015.93

    Elevated levels of Mg 2+ in APP/PS1 transgenic mice decrease the expression of IL-1β. The APP/PS1 transgenic mice at the age of 4 months were administered Mg 2+ (100 mg/kg/d) for 2 months before collecting the brain ( a ). In select experiments, the brains of APP/PS1 transgenic mice at the age of 3 months were harvested and sectioned (400 μm) using a cryostat ( b ). In separate experiments, the left cerebral ventricle was injected with Mg 2+ (2 μg/5 μl) or vehicle (PBS) and the right cerebral ventricle was injected (i.c.v.) with D1A cells, which was pre-transfected with IL-1β promoter in the right cerebral ventricle ( c ). In distinct experiments, the left cerebral ventricle was injected with Mg 2+ (2 μg/5 μl) or vehicle (PBS) before staining with IL-1β antibody and scanning under two-photon microscopy ( d ). The immunoreactivity of IL-1β was determined by immunohistochemistry using an anti-IL-1β antibody (a left panel, b). These images are representative of six independent experiments, all with similar results. IL-1β protein and mRNA levels were determined by qRT-PCR and IL-1β enzyme immunoassay kits, respectively (a right panel). The total amounts of GAPDH and protein served as an internal control. The experimental cartoon and real surgery images are shown (c, d upper panel). Luciferase activities from the different groups of mice were measured using a live animal imaging system (c lower panel). The immunofluorescence of IL-1β was scanned using a two-photon microscope (d lower panel). The data represent the means ± S.E. of three independent experiments. * p
    Figure Legend Snippet: Elevated levels of Mg 2+ in APP/PS1 transgenic mice decrease the expression of IL-1β. The APP/PS1 transgenic mice at the age of 4 months were administered Mg 2+ (100 mg/kg/d) for 2 months before collecting the brain ( a ). In select experiments, the brains of APP/PS1 transgenic mice at the age of 3 months were harvested and sectioned (400 μm) using a cryostat ( b ). In separate experiments, the left cerebral ventricle was injected with Mg 2+ (2 μg/5 μl) or vehicle (PBS) and the right cerebral ventricle was injected (i.c.v.) with D1A cells, which was pre-transfected with IL-1β promoter in the right cerebral ventricle ( c ). In distinct experiments, the left cerebral ventricle was injected with Mg 2+ (2 μg/5 μl) or vehicle (PBS) before staining with IL-1β antibody and scanning under two-photon microscopy ( d ). The immunoreactivity of IL-1β was determined by immunohistochemistry using an anti-IL-1β antibody (a left panel, b). These images are representative of six independent experiments, all with similar results. IL-1β protein and mRNA levels were determined by qRT-PCR and IL-1β enzyme immunoassay kits, respectively (a right panel). The total amounts of GAPDH and protein served as an internal control. The experimental cartoon and real surgery images are shown (c, d upper panel). Luciferase activities from the different groups of mice were measured using a live animal imaging system (c lower panel). The immunofluorescence of IL-1β was scanned using a two-photon microscope (d lower panel). The data represent the means ± S.E. of three independent experiments. * p

    Techniques Used: Transgenic Assay, Mouse Assay, Expressing, Injection, Transfection, Staining, Microscopy, Immunohistochemistry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Luciferase, Imaging, Immunofluorescence

    MgT treatment attenuated the effects of Aβ oligomers in the CSF for inducing the expression of IL-1β in the cerebral cortex. The WT C57BL/6 mice at the age of 3 months were injected (i.c.v) with Aβ oligomers (2 μg/5 μl) in the absence or presence of Mg 2+ (2 μg/5 μl). The brains were then collected and sectioned after 24 h ( a and b ). In select experiments, the brains of WT C57BL/6 mice at the age of 3 months were harvested and freshly sectioned (400 μm) before treatment with Aβ oligomers (1 μM) in the absence or presence of MgT (50 μM) for 24 h ( c ). In distinct experiments, A172 cells were treated with Aβ oligomers (1 μM) in the absence or presence of MgT (50 μM) for 24 h ( d ). The immunoreactivity of IL-1β was determined by immunohistochemistry using an anti-IL-1β antibody ( a and c ). These images are representative of six independent experiments, all with similar results. IL-1β mRNA and protein levels were determined by qRT-PCR and IL-1β enzyme immunoassay kits, respectively (b and d). The total amounts of GAPDH and protein served as an internal control. The data represent the means ± SE of three independent experiments. * P
    Figure Legend Snippet: MgT treatment attenuated the effects of Aβ oligomers in the CSF for inducing the expression of IL-1β in the cerebral cortex. The WT C57BL/6 mice at the age of 3 months were injected (i.c.v) with Aβ oligomers (2 μg/5 μl) in the absence or presence of Mg 2+ (2 μg/5 μl). The brains were then collected and sectioned after 24 h ( a and b ). In select experiments, the brains of WT C57BL/6 mice at the age of 3 months were harvested and freshly sectioned (400 μm) before treatment with Aβ oligomers (1 μM) in the absence or presence of MgT (50 μM) for 24 h ( c ). In distinct experiments, A172 cells were treated with Aβ oligomers (1 μM) in the absence or presence of MgT (50 μM) for 24 h ( d ). The immunoreactivity of IL-1β was determined by immunohistochemistry using an anti-IL-1β antibody ( a and c ). These images are representative of six independent experiments, all with similar results. IL-1β mRNA and protein levels were determined by qRT-PCR and IL-1β enzyme immunoassay kits, respectively (b and d). The total amounts of GAPDH and protein served as an internal control. The data represent the means ± SE of three independent experiments. * P

    Techniques Used: Expressing, Mouse Assay, Injection, Immunohistochemistry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Proposed cascade of the signaling events regulating the expression of IL-1β by MgT. In detail, attenuated levels of Mg 2+ in APP/PS1 transgenic mice will elevate the production of IL-1β and Aβ via ERK1/2- and PPARγ-dependent pathways in glial cells of the APP/PS1 transgenic mouse brain, which in turn will potentially regulate the pathogenesis of AD. Interestingly, the highly secreted IL-1β and Aβ in the CSF are able to regulate the synthesis of IL-1β in the cerebral cortex of APP/PS1 mice. These observations might be instrumental for understanding the roles of Mg 2+ in suppressing the neuroinflammation of AD.
    Figure Legend Snippet: Proposed cascade of the signaling events regulating the expression of IL-1β by MgT. In detail, attenuated levels of Mg 2+ in APP/PS1 transgenic mice will elevate the production of IL-1β and Aβ via ERK1/2- and PPARγ-dependent pathways in glial cells of the APP/PS1 transgenic mouse brain, which in turn will potentially regulate the pathogenesis of AD. Interestingly, the highly secreted IL-1β and Aβ in the CSF are able to regulate the synthesis of IL-1β in the cerebral cortex of APP/PS1 mice. These observations might be instrumental for understanding the roles of Mg 2+ in suppressing the neuroinflammation of AD.

    Techniques Used: Expressing, Transgenic Assay, Mouse Assay

    Involvement of ERK1/2 and PPARγ pathways in regulating the expression of IL-1β in MgT-treated A172 or D1A cells. Human glioblastoma A172 ( a ) or mouse astrocytes/microglia D1A cells ( b ) were treated with MgT (50 μM) for 48 h. In select experiments, A172 cells were treated with PD98059 (10 μM) in the absence or presence of MgT (50 μM) for 48 h ( c and e ). In separate experiments, the cells were transfected with ERK1/2 ( d ) or PPARγ siRNA ( f ) before incubation with MgT (50 μM) for 48 h. In distinct experiments, A172 cells were treated with GW9662 (1 μM) in the absence or presence of MgT (50 μM) for 48 h ( e '). In other experiments, primary cultured astrocytes were treated with MgT (50 μM) in the absence or presence of PD98059 (10 μM) (g) or GW9662 (1 μM) for 48 h (h). Total ERK1/2 ( c , d and g upper panel), phosphorylated ERK1/2 levels ( c and g upper panel), total PPARγ ( e and f upper panel), and phosphorylated PPARγ ( e ) were detected by immunoblotting using specific Abs. Equal lane loading is demonstrated by the similar intensities of total β-actin. IL-1β protein and mRNA levels were determined by IL-1β enzyme immunoassay kits and qRT-PCR, respectively. The total amounts of protein and GAPDH served as internal controls. The data represent the means ± SE of three independent experiments. * P
    Figure Legend Snippet: Involvement of ERK1/2 and PPARγ pathways in regulating the expression of IL-1β in MgT-treated A172 or D1A cells. Human glioblastoma A172 ( a ) or mouse astrocytes/microglia D1A cells ( b ) were treated with MgT (50 μM) for 48 h. In select experiments, A172 cells were treated with PD98059 (10 μM) in the absence or presence of MgT (50 μM) for 48 h ( c and e ). In separate experiments, the cells were transfected with ERK1/2 ( d ) or PPARγ siRNA ( f ) before incubation with MgT (50 μM) for 48 h. In distinct experiments, A172 cells were treated with GW9662 (1 μM) in the absence or presence of MgT (50 μM) for 48 h ( e '). In other experiments, primary cultured astrocytes were treated with MgT (50 μM) in the absence or presence of PD98059 (10 μM) (g) or GW9662 (1 μM) for 48 h (h). Total ERK1/2 ( c , d and g upper panel), phosphorylated ERK1/2 levels ( c and g upper panel), total PPARγ ( e and f upper panel), and phosphorylated PPARγ ( e ) were detected by immunoblotting using specific Abs. Equal lane loading is demonstrated by the similar intensities of total β-actin. IL-1β protein and mRNA levels were determined by IL-1β enzyme immunoassay kits and qRT-PCR, respectively. The total amounts of protein and GAPDH served as internal controls. The data represent the means ± SE of three independent experiments. * P

    Techniques Used: Expressing, Transfection, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

    Aβ oligomers in the CSF of APP/PS1 mice have the ability to stimulate the expression of IL-1β in the cerebral cortex of WT mice. The APP/PS1 Tg mice at the age of 4 months were treated with Mg 2+ (100 mg/kg/d) for 5 months before collecting the CSF and brains ( a-c ). Cerebrospinal fluid (CSF) was then injected into the wild type C57BL/6 mice in the absence or presence of Aβ antibody (1 μg/5 μl) for two weeks before scarifice ( d-f ). The production of Aβ 1-42 was determined by ELISA kits, and the total amount of protein served as an internal control ( a ). The immunoreactivity of Aβ and IL-1β was determined by immunohistochemistry using an anti-Aβ or IL-1β antibody ( c, d ). These images are representative of six independent experiments, all with similar results. The number of AP/fields was calculated according to the images of IHC ( b ). IL-1β mRNA and protein levels were determined by qRT-PCR and IL-1β enzyme immunoassay kits, respectively ( e, f ). The total amounts of GAPDH and protein served as an internal control. * p
    Figure Legend Snippet: Aβ oligomers in the CSF of APP/PS1 mice have the ability to stimulate the expression of IL-1β in the cerebral cortex of WT mice. The APP/PS1 Tg mice at the age of 4 months were treated with Mg 2+ (100 mg/kg/d) for 5 months before collecting the CSF and brains ( a-c ). Cerebrospinal fluid (CSF) was then injected into the wild type C57BL/6 mice in the absence or presence of Aβ antibody (1 μg/5 μl) for two weeks before scarifice ( d-f ). The production of Aβ 1-42 was determined by ELISA kits, and the total amount of protein served as an internal control ( a ). The immunoreactivity of Aβ and IL-1β was determined by immunohistochemistry using an anti-Aβ or IL-1β antibody ( c, d ). These images are representative of six independent experiments, all with similar results. The number of AP/fields was calculated according to the images of IHC ( b ). IL-1β mRNA and protein levels were determined by qRT-PCR and IL-1β enzyme immunoassay kits, respectively ( e, f ). The total amounts of GAPDH and protein served as an internal control. * p

    Techniques Used: Mouse Assay, Expressing, Injection, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Quantitative RT-PCR

    IL-1β was upregulated in AD patients and APP/PS1 transgenic mice. The tissue blocks of human brains at different stages of AD were collected from the New York Brain Bank at Columbia University. From 40 μm free-floating slices were prepared using a cryostat. ( a ) In select experiments, the brains of 3-month-old APP/PS1 transgenic mice were collected after anesthesia and perfusion. ( b – d ) The immunoreactivity of IL-1β was determined by immunohistochemistry using an anti-IL-1β antibody ( a and b ). These images are representative of six independent experiments, all with similar results. IL-1β mRNA levels were determined by qRT-PCR, and total amounts of GAPDH served as an internal control ( c and d upper panels). The production of IL-1β in culture medium was determined using IL-1β enzyme immunoassay kits ( c and d lower panels). The data represent the means ± SE of three independent experiments. * P
    Figure Legend Snippet: IL-1β was upregulated in AD patients and APP/PS1 transgenic mice. The tissue blocks of human brains at different stages of AD were collected from the New York Brain Bank at Columbia University. From 40 μm free-floating slices were prepared using a cryostat. ( a ) In select experiments, the brains of 3-month-old APP/PS1 transgenic mice were collected after anesthesia and perfusion. ( b – d ) The immunoreactivity of IL-1β was determined by immunohistochemistry using an anti-IL-1β antibody ( a and b ). These images are representative of six independent experiments, all with similar results. IL-1β mRNA levels were determined by qRT-PCR, and total amounts of GAPDH served as an internal control ( c and d upper panels). The production of IL-1β in culture medium was determined using IL-1β enzyme immunoassay kits ( c and d lower panels). The data represent the means ± SE of three independent experiments. * P

    Techniques Used: Transgenic Assay, Mouse Assay, Immunohistochemistry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    MgT treatment diminished the effects of IL-1β in the CSF with respect to inducing the expression of IL-1β in the cerebral cortex. WT C57BL/6 mice at the age of 3 months were intracerebroventricularly injected with IL-1β (0.5 μg/5 μl) in the absence or presence of Mg 2+ (2 μg/5 μl). The brains were then collected and sectioned after 24 h ( a and b ). In select experiments, the brains of WT C57BL/6 mice at the age of 3 months were harvested and freshly sectioned (400 μm) before treatment with IL-1β (100 ng ml –1 ) in the absence or presence of MgT (50 μM) for 24 h ( c ). In separate experiments, A172 cells were treated with IL-1β (100 ng ml –1 ) in the absence or presence of MgT (50 μM) for 24 h ( d ). The immunoreactivity of IL-1β was determined by immunohistochemistry using an anti-IL-1β or -APH-1 antibody ( a and c ). These images are representative of six independent experiments, all with similar results. IL-1β mRNA and protein levels were determined by qRT-PCR and IL-1β enzyme immunoassay kits, respectively ( b and d ). The total amounts of GAPDH and protein served as an internal control. The data represent the means ± SE of three independent experiments. * P
    Figure Legend Snippet: MgT treatment diminished the effects of IL-1β in the CSF with respect to inducing the expression of IL-1β in the cerebral cortex. WT C57BL/6 mice at the age of 3 months were intracerebroventricularly injected with IL-1β (0.5 μg/5 μl) in the absence or presence of Mg 2+ (2 μg/5 μl). The brains were then collected and sectioned after 24 h ( a and b ). In select experiments, the brains of WT C57BL/6 mice at the age of 3 months were harvested and freshly sectioned (400 μm) before treatment with IL-1β (100 ng ml –1 ) in the absence or presence of MgT (50 μM) for 24 h ( c ). In separate experiments, A172 cells were treated with IL-1β (100 ng ml –1 ) in the absence or presence of MgT (50 μM) for 24 h ( d ). The immunoreactivity of IL-1β was determined by immunohistochemistry using an anti-IL-1β or -APH-1 antibody ( a and c ). These images are representative of six independent experiments, all with similar results. IL-1β mRNA and protein levels were determined by qRT-PCR and IL-1β enzyme immunoassay kits, respectively ( b and d ). The total amounts of GAPDH and protein served as an internal control. The data represent the means ± SE of three independent experiments. * P

    Techniques Used: Expressing, Mouse Assay, Injection, Immunohistochemistry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    16) Product Images from "Carbon Monoxide Blocks Lipopolysaccharide-Induced Gene Expression by Interfering with Proximal TLR4 to NF-?B Signal Transduction in Human Monocytes"

    Article Title: Carbon Monoxide Blocks Lipopolysaccharide-Induced Gene Expression by Interfering with Proximal TLR4 to NF-?B Signal Transduction in Human Monocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0008139

    CO suppression of LPS-induced IL-1β transcription, mRNA expression, and protein production. (A) Nuclear run-on assays in THP-1 cells (2×10 7 ) showed that CO represses IL-1β transcription by almost 98% within 30 min of LPS stimulation (p = 0.001) and the effect decreased to 67% at 60 min (p = 0.01). (B) A time course analysis of IL-1β mRNA expression in THP-1 cells (2×10 6 ) by qRT-PCR showed that CO strongly suppressed LPS-induced IL-1β mRNA by more than 90% within 30 min of exposure (p
    Figure Legend Snippet: CO suppression of LPS-induced IL-1β transcription, mRNA expression, and protein production. (A) Nuclear run-on assays in THP-1 cells (2×10 7 ) showed that CO represses IL-1β transcription by almost 98% within 30 min of LPS stimulation (p = 0.001) and the effect decreased to 67% at 60 min (p = 0.01). (B) A time course analysis of IL-1β mRNA expression in THP-1 cells (2×10 6 ) by qRT-PCR showed that CO strongly suppressed LPS-induced IL-1β mRNA by more than 90% within 30 min of exposure (p

    Techniques Used: Expressing, Quantitative RT-PCR

    CO downregulation of LPS-induced IL-1β and IL-8 protein. CO downregulated LPS-induced production of (A) IL-1β and (B) IL-8 in THP-1 cells (p
    Figure Legend Snippet: CO downregulation of LPS-induced IL-1β and IL-8 protein. CO downregulated LPS-induced production of (A) IL-1β and (B) IL-8 in THP-1 cells (p

    Techniques Used:

    Effects of CO on LPS-induced IL-1β production in the presence or absence of stress kinase inhibitors. THP-1 cells (2×10 6 ) were pretreated with inhibitors of (A) p38 MAPK (SB202190; 0.1 µM), (B) ERK1/2 (PD98059; 30 µM), (C) JNK (Inhibitor II; 10 µM), and (D) Akt (LY294002; 10 µM) for 30 min and then stimulated with LPS (1 µg/ml) in the presence or absence of CO gas (250 ppm) for 2 h. DMSO (0.1%) vehicle was used as control. IL-1β production was measured in supernatants by ELISA. Results are mean ± SEM of three to five independent experiments.
    Figure Legend Snippet: Effects of CO on LPS-induced IL-1β production in the presence or absence of stress kinase inhibitors. THP-1 cells (2×10 6 ) were pretreated with inhibitors of (A) p38 MAPK (SB202190; 0.1 µM), (B) ERK1/2 (PD98059; 30 µM), (C) JNK (Inhibitor II; 10 µM), and (D) Akt (LY294002; 10 µM) for 30 min and then stimulated with LPS (1 µg/ml) in the presence or absence of CO gas (250 ppm) for 2 h. DMSO (0.1%) vehicle was used as control. IL-1β production was measured in supernatants by ELISA. Results are mean ± SEM of three to five independent experiments.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    17) Product Images from "Alcohol intake aggravates adipose browning and muscle atrophy in cancer-associated cachexia"

    Article Title: Alcohol intake aggravates adipose browning and muscle atrophy in cancer-associated cachexia

    Journal: Oncotarget

    doi: 10.18632/oncotarget.22243

    Alcohol promoted apoptosis and inflammation in skeletal muscle of tumor mice (A-C) Pro-caspase 1, activated caspase 1, Pro-caspase 3 and activated caspase 3 protein contents in GA muscle. (D-F) NLRP3 and IL-1β protein contents in GA muscle. ( * p
    Figure Legend Snippet: Alcohol promoted apoptosis and inflammation in skeletal muscle of tumor mice (A-C) Pro-caspase 1, activated caspase 1, Pro-caspase 3 and activated caspase 3 protein contents in GA muscle. (D-F) NLRP3 and IL-1β protein contents in GA muscle. ( * p

    Techniques Used: Mouse Assay

    18) Product Images from "Modulation of calcium signaling pathway by hepatitis C virus core protein stimulates NLRP3 inflammasome activation"

    Article Title: Modulation of calcium signaling pathway by hepatitis C virus core protein stimulates NLRP3 inflammasome activation

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007593

    Component(s) within the incoming HCV virion stimulate NLRP3 inflammasome activation and IL-1β production. (A) IL-1β ELISA in THP-1 cells. THP-1 cells were differentiated with PMA overnight then rested for another 24hrs. The cells were exposed to infectious supernatant containing live-HCV or HCV inactivated by ultraviolet irradiation (uv-HCV) then the response was monitored. (B) immunoblot showing the replication capacity of uv-HCV as compared to live-HCV. (C) Differentiated THP-1 cells were exposed to infectious supernatant containing HCV inactivated by ultraviolet irradiation (uv-HCV) then the response was monitored over the indicated time course. (D) immunoblot showing the processing of caspase-1 post exposure to uv-HCV. Actin serves as a loading control. (E) ASC-specks in uv-inactivated HCV or live-HCV treated THP-1 cells. Differentiated THP-1 cells were stimulated with uv-HCV/live-HCV for 1hr then fixed with paraformaldehyde. Fixed cells were stained with anti-ASC antibody and DAPI. Red stains ASC-specks and DAPI stains the nuclei. ASC specks are indicated by the arrows. Experiments were performed with at least two technical replicates and are representative of three independent experiments. Data are presented as means +/- SD. *P
    Figure Legend Snippet: Component(s) within the incoming HCV virion stimulate NLRP3 inflammasome activation and IL-1β production. (A) IL-1β ELISA in THP-1 cells. THP-1 cells were differentiated with PMA overnight then rested for another 24hrs. The cells were exposed to infectious supernatant containing live-HCV or HCV inactivated by ultraviolet irradiation (uv-HCV) then the response was monitored. (B) immunoblot showing the replication capacity of uv-HCV as compared to live-HCV. (C) Differentiated THP-1 cells were exposed to infectious supernatant containing HCV inactivated by ultraviolet irradiation (uv-HCV) then the response was monitored over the indicated time course. (D) immunoblot showing the processing of caspase-1 post exposure to uv-HCV. Actin serves as a loading control. (E) ASC-specks in uv-inactivated HCV or live-HCV treated THP-1 cells. Differentiated THP-1 cells were stimulated with uv-HCV/live-HCV for 1hr then fixed with paraformaldehyde. Fixed cells were stained with anti-ASC antibody and DAPI. Red stains ASC-specks and DAPI stains the nuclei. ASC specks are indicated by the arrows. Experiments were performed with at least two technical replicates and are representative of three independent experiments. Data are presented as means +/- SD. *P

    Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, Irradiation, Staining

    HCV capsid protein and ion-channel p7, but not envelope proteins (E1 and E2) activate NLRP3 inflammasome signaling. (A) IL-1β ELISA (top panel) in U2OS cells reconstituted with NLRP3 inflammasome components. Human proIL-1β, procasp1, ASC and NLRP3 were co-transfected with constructs expressing vector only or HCV core or HCV-p7 or HCV-E1E2. Lower panel of (A) is a western blot depicting the expression of each transfected constructs including the loading control, actin. (B) Relative luciferase activity measured in hepatoma Huh7 cells post infection with HCVpp (upper panel). Lower panel (B), IL-1β ELISA in THP-1 cells stimulated with (HCVpp) or VSVpp. Experiments were performed with replicates and are representative of at least two independent experiments. Data are presented as means +/- SD. *P
    Figure Legend Snippet: HCV capsid protein and ion-channel p7, but not envelope proteins (E1 and E2) activate NLRP3 inflammasome signaling. (A) IL-1β ELISA (top panel) in U2OS cells reconstituted with NLRP3 inflammasome components. Human proIL-1β, procasp1, ASC and NLRP3 were co-transfected with constructs expressing vector only or HCV core or HCV-p7 or HCV-E1E2. Lower panel of (A) is a western blot depicting the expression of each transfected constructs including the loading control, actin. (B) Relative luciferase activity measured in hepatoma Huh7 cells post infection with HCVpp (upper panel). Lower panel (B), IL-1β ELISA in THP-1 cells stimulated with (HCVpp) or VSVpp. Experiments were performed with replicates and are representative of at least two independent experiments. Data are presented as means +/- SD. *P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Transfection, Construct, Expressing, Plasmid Preparation, Western Blot, Luciferase, Activity Assay, Infection

    Full length of domain-I of HCV core protein is required for optimal NLRP3 inflammasome activation. (A) A schematic of HCV core protein major domains. (B) ELISA of IL-1β in the reconstituted cells transfected with various c-terminus truncation mutants of HCV core protein. Anti-Flag antibody stains the core constructs and actin serves as a loading control. Experiments were performed with three replicates and are representative of three independent experiments. Data are represented as means and +/- SD, *P
    Figure Legend Snippet: Full length of domain-I of HCV core protein is required for optimal NLRP3 inflammasome activation. (A) A schematic of HCV core protein major domains. (B) ELISA of IL-1β in the reconstituted cells transfected with various c-terminus truncation mutants of HCV core protein. Anti-Flag antibody stains the core constructs and actin serves as a loading control. Experiments were performed with three replicates and are representative of three independent experiments. Data are represented as means and +/- SD, *P

    Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, Transfection, Construct

    HCV core protein efficiently mediates NLRP3 activation and IL-1β release in the reconstituted system. (A) ELISA of IL-1β in U2OS cells reconstituted with NLRP3 inflammasome components. Top panel of (A) shows ELISA in vector or core or Flu-M2 transfected cells. Lower panel of (A) is IP-western blot depicting the expression of each NLRP3 inflammasome components (ASC, procasp-1, proIL-1β and NLRP3) and the co-transfected vector or core or Flu-M2. (B) ELISA (upper panel) in U2OS cells reconstituted with NLRP3 inflammasome components and transfected with a decreasing doses of HCV core protein expressing construct (western blot, lower panel). (C) ELISA revealing increase in IL-1β release with increasing input of proIL-1β expressing construct (upper panel) and input protein (lower panel) in the reconstituted cells. (D) ASC-specks in vector or core expressing reconstituted cells. Green stains HCV core protein, red stains ASC-specks and blue stains the nuclei. Experiments were performed with more than two replicates and are representative of three independent experiments. Data are represented as means +/- SD. For (A) *P = 0.0113. For (B) *P
    Figure Legend Snippet: HCV core protein efficiently mediates NLRP3 activation and IL-1β release in the reconstituted system. (A) ELISA of IL-1β in U2OS cells reconstituted with NLRP3 inflammasome components. Top panel of (A) shows ELISA in vector or core or Flu-M2 transfected cells. Lower panel of (A) is IP-western blot depicting the expression of each NLRP3 inflammasome components (ASC, procasp-1, proIL-1β and NLRP3) and the co-transfected vector or core or Flu-M2. (B) ELISA (upper panel) in U2OS cells reconstituted with NLRP3 inflammasome components and transfected with a decreasing doses of HCV core protein expressing construct (western blot, lower panel). (C) ELISA revealing increase in IL-1β release with increasing input of proIL-1β expressing construct (upper panel) and input protein (lower panel) in the reconstituted cells. (D) ASC-specks in vector or core expressing reconstituted cells. Green stains HCV core protein, red stains ASC-specks and blue stains the nuclei. Experiments were performed with more than two replicates and are representative of three independent experiments. Data are represented as means +/- SD. For (A) *P = 0.0113. For (B) *P

    Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Transfection, Western Blot, Expressing, Construct

    HCV core protein induces IL-1β release in macrophages. (A) IL-1β ELISA in THP-1 cells stably expressing vector only or HCV-core or Flu-M2 post differentiation with PMA. To prime and stimulate proIL-1β expression, cells were stimulated with either recombinant TNF (rTNF) or in (B) transfected with HCV polyU/UC RNA PAMP. Lower panel of (A) and (B) show the expression of core or Flu-M2 in the stable cell lines. (C) ASC-specks in THP-1 cells treated with recombinant core (rHCV-core) or recombinant GFP (rGFP). Red stains ASC-specks, green stains core or rGFP, and blue stains nuclei. ASC-specks are denoted by the white arrows. (D) ELISA showing the release of IL-1β in TNF-primed THP-1 treated with recombinant core (rHCV-core) or ATP. Top panel of (D) shows the release of IL-1β in supernatant and lower panel of (D) shows by western blot cleaved IL-1β protein in each treatment condition. (E) Primary human monocyte-derived macrophages were primed with rTNF for signal-one then treated with rHCV-Core or Ng. Upper panel of (E) is IL-1β ELISA and the lower panel shows production of cleaved of IL-1β both in supernatant and lysate. (F) ELISA of IL-1β in THP-1 non-targeting control cells or THP1 cells lacking NLRP3. These cells were primed with rTNF then stimulated with rHCV-core. (G) western blot depicting the production or lack of cleaved IL-1β in NLRP3 knockout or non-targeting control (NC) cells. Experiments were performed with two replicates and are representative of three independent experiments. Data are represented as means and +/- SD. For (A) *P = 0.0228 and *P = 0.0398, for (B) *P = 0.0317 and *P = 0.0495, for (D) *P = 0.0114, for (E) *P = 0.0178, for (F) ***p
    Figure Legend Snippet: HCV core protein induces IL-1β release in macrophages. (A) IL-1β ELISA in THP-1 cells stably expressing vector only or HCV-core or Flu-M2 post differentiation with PMA. To prime and stimulate proIL-1β expression, cells were stimulated with either recombinant TNF (rTNF) or in (B) transfected with HCV polyU/UC RNA PAMP. Lower panel of (A) and (B) show the expression of core or Flu-M2 in the stable cell lines. (C) ASC-specks in THP-1 cells treated with recombinant core (rHCV-core) or recombinant GFP (rGFP). Red stains ASC-specks, green stains core or rGFP, and blue stains nuclei. ASC-specks are denoted by the white arrows. (D) ELISA showing the release of IL-1β in TNF-primed THP-1 treated with recombinant core (rHCV-core) or ATP. Top panel of (D) shows the release of IL-1β in supernatant and lower panel of (D) shows by western blot cleaved IL-1β protein in each treatment condition. (E) Primary human monocyte-derived macrophages were primed with rTNF for signal-one then treated with rHCV-Core or Ng. Upper panel of (E) is IL-1β ELISA and the lower panel shows production of cleaved of IL-1β both in supernatant and lysate. (F) ELISA of IL-1β in THP-1 non-targeting control cells or THP1 cells lacking NLRP3. These cells were primed with rTNF then stimulated with rHCV-core. (G) western blot depicting the production or lack of cleaved IL-1β in NLRP3 knockout or non-targeting control (NC) cells. Experiments were performed with two replicates and are representative of three independent experiments. Data are represented as means and +/- SD. For (A) *P = 0.0228 and *P = 0.0398, for (B) *P = 0.0317 and *P = 0.0495, for (D) *P = 0.0114, for (E) *P = 0.0178, for (F) ***p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Stable Transfection, Expressing, Plasmid Preparation, Recombinant, Transfection, Western Blot, Derivative Assay, Knock-Out

    HCV Core-mediated IL-1β production in macrophages. A working model of how HCV-mediated inflammation is propagated by HCV core protein in macrophages. Macrophages priming by TNF and/or viral PAMPs such as viral RNA leads to signal-one induction via NFκB mediated signaling. Macrophage engulfment of HCV leads to core accumulation in the cytoplasm, which stimulates the activation of the NLRP3 inflammasome. Core triggered NLRP3 activation is induced by HCV-core-mediated calcium mobilization through phospholipase C-dependent pathways.
    Figure Legend Snippet: HCV Core-mediated IL-1β production in macrophages. A working model of how HCV-mediated inflammation is propagated by HCV core protein in macrophages. Macrophages priming by TNF and/or viral PAMPs such as viral RNA leads to signal-one induction via NFκB mediated signaling. Macrophage engulfment of HCV leads to core accumulation in the cytoplasm, which stimulates the activation of the NLRP3 inflammasome. Core triggered NLRP3 activation is induced by HCV-core-mediated calcium mobilization through phospholipase C-dependent pathways.

    Techniques Used: Activation Assay

    Core from HCV infected patients variably stimulate NLRP3 inflammasome activation. (A) IL-1β ELISA. HCV core, from acutely infected individuals, expressing constructs co-transfected with NLRP3 inflammasome components. (B) western blot depicting the expression of the patient-derived core as well as the inflammasome components. Experiments were performed with three replicates and are representative of four independent experiments. Data are represented as means and +/- SD, *P
    Figure Legend Snippet: Core from HCV infected patients variably stimulate NLRP3 inflammasome activation. (A) IL-1β ELISA. HCV core, from acutely infected individuals, expressing constructs co-transfected with NLRP3 inflammasome components. (B) western blot depicting the expression of the patient-derived core as well as the inflammasome components. Experiments were performed with three replicates and are representative of four independent experiments. Data are represented as means and +/- SD, *P

    Techniques Used: Infection, Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Construct, Transfection, Western Blot, Derivative Assay

    HCV core protein modulates cellular calcium to stimulate inflammasome activation and IL-1β release. (A) Wheat germ agglutinin staining of THP-1 cells treated with HCV. THP-1 cells were exposed to HCV 30-60minutes. Before fixing with paraformaldehyde, cells were labeled with wheat germ agglutinin to mark the plasma membrane. Red stains the plasma membrane (wheat germ agglutinin), green stains HCV core protein within THP-1 cytoplasm and blue (DAPI) stains the nuclei. (B) Calcium flux in THP-1 cells post stimulation with rHCV-Core or ionomycin or rGFP. (C) immunoblot showing the presence and absence of cleaved IL-1β and caspase-1 in HCV treated cells in the presence of u-73343 (inactive phospholipase C inhibitor) or u-73122 (active phospholipase C inhibitor, control) or DMSO (vehicle control). (D) ELISA of IL-1β in THP-1 cells post stimulation with HCV polyU/UC and rHCV-core or HCV polyU/UC and Ng in the presence of u-73122 or control u-73343. (E) TNF primed THP-1 cells were treated with rHCV-core or Ng in the presence and absence of calcium inhibitor (u-73122) or inactive analog (u-73433) or vehicle control (DMSO). Lower panel of (E) shows immunoblot of active IL-1β protein post treatment with calcium inhibitor. Experiments were performed in duplicates and are representative of three or more independent experiments. Data are represented as means and +/- SD, for (D) and (E) *P
    Figure Legend Snippet: HCV core protein modulates cellular calcium to stimulate inflammasome activation and IL-1β release. (A) Wheat germ agglutinin staining of THP-1 cells treated with HCV. THP-1 cells were exposed to HCV 30-60minutes. Before fixing with paraformaldehyde, cells were labeled with wheat germ agglutinin to mark the plasma membrane. Red stains the plasma membrane (wheat germ agglutinin), green stains HCV core protein within THP-1 cytoplasm and blue (DAPI) stains the nuclei. (B) Calcium flux in THP-1 cells post stimulation with rHCV-Core or ionomycin or rGFP. (C) immunoblot showing the presence and absence of cleaved IL-1β and caspase-1 in HCV treated cells in the presence of u-73343 (inactive phospholipase C inhibitor) or u-73122 (active phospholipase C inhibitor, control) or DMSO (vehicle control). (D) ELISA of IL-1β in THP-1 cells post stimulation with HCV polyU/UC and rHCV-core or HCV polyU/UC and Ng in the presence of u-73122 or control u-73343. (E) TNF primed THP-1 cells were treated with rHCV-core or Ng in the presence and absence of calcium inhibitor (u-73122) or inactive analog (u-73433) or vehicle control (DMSO). Lower panel of (E) shows immunoblot of active IL-1β protein post treatment with calcium inhibitor. Experiments were performed in duplicates and are representative of three or more independent experiments. Data are represented as means and +/- SD, for (D) and (E) *P

    Techniques Used: Activation Assay, Staining, Labeling, Enzyme-linked Immunosorbent Assay

    19) Product Images from "Activation of Oxytocin Neurons Improves Cardiac Function in a Pressure-Overload Model of Heart Failure"

    Article Title: Activation of Oxytocin Neurons Improves Cardiac Function in a Pressure-Overload Model of Heart Failure

    Journal: JACC: Basic to Translational Science

    doi: 10.1016/j.jacbts.2020.03.007

    Hearts From TAC Animals With PVN OXT Neuron Activation Had Improved LV Function and Less Collagen III and IL-1β Expression Compared with Untreated TAC Animals (A) Langendorff studies of excised hearts revealed that LVDP was significantly impaired in TAC animals (n = 5) compared with both Sham (n = 8) and PVN OXT treatment animals (n = 8). Contractility (B) and relaxation (C) measurements were calculated using the derivative of the LV pressure wave; both were significantly compromised in TAC animals (n = 5) compared with Sham animals (n = 8), whereas TAC + OXT animals (n = 8) did not significantly deviate from Sham. (D) Western-blot analysis revealed significant elevations in cardiac levels of IL-1β in TAC animals (n = 7) compared with PVN OXT treatment animals (n = 8) using a Student’s t -test, as Sham levels were negligible; IL-1β quantitation is relative to GAPDH. (E) Western-blot assays also revealed significant elevations in cardiac levels of the fibrosis marker, collagen III, in TAC animals (n = 7) compared with both Sham (n = 7) and PVN OXT-treated animals (n = 9); collagen III quantitation is relative to GAPDH and to Sham values. (F) Trichrome-stained histological sections indicate increased fibrosis in all disease groups compared with Sham, as well as greater right-ventricular wall thinning in TAC-untreated animals compared with PVN OXT-treated animals. Data were analyzed using a 1-way analysis of variance with Tukey’s post hoc test; ∗p
    Figure Legend Snippet: Hearts From TAC Animals With PVN OXT Neuron Activation Had Improved LV Function and Less Collagen III and IL-1β Expression Compared with Untreated TAC Animals (A) Langendorff studies of excised hearts revealed that LVDP was significantly impaired in TAC animals (n = 5) compared with both Sham (n = 8) and PVN OXT treatment animals (n = 8). Contractility (B) and relaxation (C) measurements were calculated using the derivative of the LV pressure wave; both were significantly compromised in TAC animals (n = 5) compared with Sham animals (n = 8), whereas TAC + OXT animals (n = 8) did not significantly deviate from Sham. (D) Western-blot analysis revealed significant elevations in cardiac levels of IL-1β in TAC animals (n = 7) compared with PVN OXT treatment animals (n = 8) using a Student’s t -test, as Sham levels were negligible; IL-1β quantitation is relative to GAPDH. (E) Western-blot assays also revealed significant elevations in cardiac levels of the fibrosis marker, collagen III, in TAC animals (n = 7) compared with both Sham (n = 7) and PVN OXT-treated animals (n = 9); collagen III quantitation is relative to GAPDH and to Sham values. (F) Trichrome-stained histological sections indicate increased fibrosis in all disease groups compared with Sham, as well as greater right-ventricular wall thinning in TAC-untreated animals compared with PVN OXT-treated animals. Data were analyzed using a 1-way analysis of variance with Tukey’s post hoc test; ∗p

    Techniques Used: Activation Assay, Expressing, Western Blot, Quantitation Assay, Marker, Staining

    20) Product Images from "Globular Adiponectin Inhibits Lipopolysaccharide-Primed Inflammasomes Activation in Macrophages via Autophagy Induction: The Critical Role of AMPK Signaling"

    Article Title: Globular Adiponectin Inhibits Lipopolysaccharide-Primed Inflammasomes Activation in Macrophages via Autophagy Induction: The Critical Role of AMPK Signaling

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18061275

    Role of autophagy induction in the suppression of IL-1β maturation and secretion by globular adiponectin in macrophages. ( A , B ) Macrophages were treated with gAcrp (0.1 μg/mL) for 18 h. ATG5 ( A ) and LC3 ( B ) expression levels were determined by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control. ( C – E ) Cells were pretreated with gAcrp (0.1 μg/mL) for 18 h in the absence or presence of 3-MA or Bafilomycin, stimulated with LPS (100 ng/mL) for 8 h, and then treated with ATP (5 mM) for 1 h; ( C ) total cellular lysates were prepared, and the levels of pro- and mature active IL-1β were measured by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control; ( D ) cell culture media were collected, and the levels of mature active IL-1β were measured by Western blot analysis. Quantitative analysis of active IL-1β (p17) was performed by densitometric analysis and shown in the lower panel. Values presented are fold change compared to LPS and ATP treatment and expressed as mean ± SEM ( n = 3). * p
    Figure Legend Snippet: Role of autophagy induction in the suppression of IL-1β maturation and secretion by globular adiponectin in macrophages. ( A , B ) Macrophages were treated with gAcrp (0.1 μg/mL) for 18 h. ATG5 ( A ) and LC3 ( B ) expression levels were determined by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control. ( C – E ) Cells were pretreated with gAcrp (0.1 μg/mL) for 18 h in the absence or presence of 3-MA or Bafilomycin, stimulated with LPS (100 ng/mL) for 8 h, and then treated with ATP (5 mM) for 1 h; ( C ) total cellular lysates were prepared, and the levels of pro- and mature active IL-1β were measured by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control; ( D ) cell culture media were collected, and the levels of mature active IL-1β were measured by Western blot analysis. Quantitative analysis of active IL-1β (p17) was performed by densitometric analysis and shown in the lower panel. Values presented are fold change compared to LPS and ATP treatment and expressed as mean ± SEM ( n = 3). * p

    Techniques Used: Expressing, Western Blot, Cell Culture

    Effects of globular adiponectin on maturation and secretion of IL-1β stimulated by lipopolysaccharide (LPS) in murine peritoneal macrophages. ( A ) Macrophages were isolated from murine peritonea, pretreated with the indicated concentrations of gAcrp for 18 h, and stimulated with LPS (100 ng/mL) for 8 h and then ATP (5 mM) for 1 h. Total cellular lysates were prepared, and the levels of pro- and mature active IL-1β were determined by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control; ( B ) after treatment with gAcrp, LPS, and ATP, as in Figure 1 A, media were collected, and the levels of mature active IL-1β were measured by Western blot analysis. Quantitative analysis of active IL-1β expression was performed by densitometric analysis and shown in the lower panel ( A , B ). Values presented are fold change compared to the cells treated with LPS and ATP and expressed as mean ± SEM ( n = 3 for A , n = 2 for B ). * p
    Figure Legend Snippet: Effects of globular adiponectin on maturation and secretion of IL-1β stimulated by lipopolysaccharide (LPS) in murine peritoneal macrophages. ( A ) Macrophages were isolated from murine peritonea, pretreated with the indicated concentrations of gAcrp for 18 h, and stimulated with LPS (100 ng/mL) for 8 h and then ATP (5 mM) for 1 h. Total cellular lysates were prepared, and the levels of pro- and mature active IL-1β were determined by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control; ( B ) after treatment with gAcrp, LPS, and ATP, as in Figure 1 A, media were collected, and the levels of mature active IL-1β were measured by Western blot analysis. Quantitative analysis of active IL-1β expression was performed by densitometric analysis and shown in the lower panel ( A , B ). Values presented are fold change compared to the cells treated with LPS and ATP and expressed as mean ± SEM ( n = 3 for A , n = 2 for B ). * p

    Techniques Used: Isolation, Western Blot, Expressing

    Proposed model for the suppression of lipopolysaccharide (LPS)-primed inflammasomes activation by globular adiponectin via autophagy induction in macrophages. Treatment with globular adiponectin induces activation of an autophagic process via 5′AMP-activated protein kinase ( AMPK ) signaling in murine macrophages. The detailed mechanisms underlying p62 induction by globular adiponectin in the context of AMPK signaling remain to be determined. LPS treatment has been shown to enhance expression of pro-IL-1β through TLR4 and NF-κB signaling. Extracellular ATP triggers the inflammasome activation through a series of biological processes leading to the cleavage and activation of pro-caspase-1, which further induces maturation and secretion of IL-1β and pyroptosis in immune cells. In this study, we clearly showed that globular adiponectin suppresses LPS-stimulated IL-1β maturation and pyroptosis via modulation of inflammasomes activation. Importantly, autophagy induction plays a crucial role in the modulation of inflammasomes activation by adiponectin. It is likely that adiponectin-induced autophagy activation regulates inflammasomes activation through inhibition of ASC speck formation and inflammasomes assembly. Furthermore, AMPK signaling plays a pivotal role in autophagy activation and prevention of inflammasomes activation by globular adiponectin. P2X7 receptor: Purinergic ATP receptor; PYD: PYRIN-PAAD-DAPIN domain; CARD: Caspase activation and recruitment domain.
    Figure Legend Snippet: Proposed model for the suppression of lipopolysaccharide (LPS)-primed inflammasomes activation by globular adiponectin via autophagy induction in macrophages. Treatment with globular adiponectin induces activation of an autophagic process via 5′AMP-activated protein kinase ( AMPK ) signaling in murine macrophages. The detailed mechanisms underlying p62 induction by globular adiponectin in the context of AMPK signaling remain to be determined. LPS treatment has been shown to enhance expression of pro-IL-1β through TLR4 and NF-κB signaling. Extracellular ATP triggers the inflammasome activation through a series of biological processes leading to the cleavage and activation of pro-caspase-1, which further induces maturation and secretion of IL-1β and pyroptosis in immune cells. In this study, we clearly showed that globular adiponectin suppresses LPS-stimulated IL-1β maturation and pyroptosis via modulation of inflammasomes activation. Importantly, autophagy induction plays a crucial role in the modulation of inflammasomes activation by adiponectin. It is likely that adiponectin-induced autophagy activation regulates inflammasomes activation through inhibition of ASC speck formation and inflammasomes assembly. Furthermore, AMPK signaling plays a pivotal role in autophagy activation and prevention of inflammasomes activation by globular adiponectin. P2X7 receptor: Purinergic ATP receptor; PYD: PYRIN-PAAD-DAPIN domain; CARD: Caspase activation and recruitment domain.

    Techniques Used: Activation Assay, Expressing, Inhibition

    Role of 5′AMP-activated protein kinase ( AMPK ) signaling in autophagy induction and suppression of IL-1β maturation by globular adiponectin. ( A ) Peritoneal macrophages were pretreated with gAcrp (0.1 μg/mL) for 24 h, followed by stimulation with LPS (100 ng/mL) for additional 30 min. Phosphorylation of AMPK was determined by Western blot analysis. Images are representative of three independent experiments showing similar results; ( B , C ) macrophages were treated with gAcrp (0.1 μg/mL) for 24 h in the absence or presence of compound C. ATG5 ( B ) and LC3 ( C ) expression levels were assessed by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control; ( D ) cells were pretreated with gAcrp (0.1 μg/mL) for 24 h in the absence or presence of compound C, stimulated with LPS (100 ng/mL) for 6 h, and then treated with ATP (5 mM) for 30 min. Levels of pro- and mature active forms of IL-1β were determined by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control.
    Figure Legend Snippet: Role of 5′AMP-activated protein kinase ( AMPK ) signaling in autophagy induction and suppression of IL-1β maturation by globular adiponectin. ( A ) Peritoneal macrophages were pretreated with gAcrp (0.1 μg/mL) for 24 h, followed by stimulation with LPS (100 ng/mL) for additional 30 min. Phosphorylation of AMPK was determined by Western blot analysis. Images are representative of three independent experiments showing similar results; ( B , C ) macrophages were treated with gAcrp (0.1 μg/mL) for 24 h in the absence or presence of compound C. ATG5 ( B ) and LC3 ( C ) expression levels were assessed by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control; ( D ) cells were pretreated with gAcrp (0.1 μg/mL) for 24 h in the absence or presence of compound C, stimulated with LPS (100 ng/mL) for 6 h, and then treated with ATP (5 mM) for 30 min. Levels of pro- and mature active forms of IL-1β were determined by Western blot analysis. Representative images from three independent experiments are shown along with β-actin as an internal loading control.

    Techniques Used: Western Blot, Expressing

    21) Product Images from "POH1 deubiquitinates pro-interleukin-1β and restricts inflammasome activity"

    Article Title: POH1 deubiquitinates pro-interleukin-1β and restricts inflammasome activity

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06455-z

    POH1 negatively modifies K63-linked polyubiquitination of pro-IL-1β. a BMDMs stimulated with LPS for the indicated time periods were immunoprecipitated (IP) with an anti-POH1 or control IgG antibody and immunoblotted (IB) with the indicated antibodies. b HEK293T cells transfected with His-POH1 and either Flag-NLRP3, Flag-caspase-1 or Flag-ASC were subjected to IP with an anti-Flag antibody and IB with an anti-His antibody. c The lysates of HEK293T cells expressing Flag-POH1 and His-pro-IL-1β were IP with an anti-Flag antibody and IB with an anti-His antibody in cell lysates. d HEK293T cells transfected with Vsv-ASC, V5-tagged pro-caspase-1, HA-tagged K48-only or K63-only Ub and His-tagged pro-IL-1β, along with or without Flag-tagged POH1, were subjected to IP with an anti-V5 antibody or anti-His antibody and then IB with the indicated antibodies. e Poh1 Δ/+ and Poh1 Δ/Δ BMDMs treated as indicated were subjected to IP with an anti-pro-IL-1β antibody and IB with the indicated antibodies. f HEK293T cells were transfected with Flag-caspase-1, Vsv-ASC, His-pro-IL-1β, Flag-POH1 (WT) or Flag-H113Q-POH1, along with HA-tagged K63-only Ub, then cells were subjected to IP with an anti-His or control IgG antibody and IB with the indicated antibodies. Similar results were obtained from three independent experiments
    Figure Legend Snippet: POH1 negatively modifies K63-linked polyubiquitination of pro-IL-1β. a BMDMs stimulated with LPS for the indicated time periods were immunoprecipitated (IP) with an anti-POH1 or control IgG antibody and immunoblotted (IB) with the indicated antibodies. b HEK293T cells transfected with His-POH1 and either Flag-NLRP3, Flag-caspase-1 or Flag-ASC were subjected to IP with an anti-Flag antibody and IB with an anti-His antibody. c The lysates of HEK293T cells expressing Flag-POH1 and His-pro-IL-1β were IP with an anti-Flag antibody and IB with an anti-His antibody in cell lysates. d HEK293T cells transfected with Vsv-ASC, V5-tagged pro-caspase-1, HA-tagged K48-only or K63-only Ub and His-tagged pro-IL-1β, along with or without Flag-tagged POH1, were subjected to IP with an anti-V5 antibody or anti-His antibody and then IB with the indicated antibodies. e Poh1 Δ/+ and Poh1 Δ/Δ BMDMs treated as indicated were subjected to IP with an anti-pro-IL-1β antibody and IB with the indicated antibodies. f HEK293T cells were transfected with Flag-caspase-1, Vsv-ASC, His-pro-IL-1β, Flag-POH1 (WT) or Flag-H113Q-POH1, along with HA-tagged K63-only Ub, then cells were subjected to IP with an anti-His or control IgG antibody and IB with the indicated antibodies. Similar results were obtained from three independent experiments

    Techniques Used: Immunoprecipitation, Transfection, Expressing

    Pro-IL-1β maturation is regulated by POH1-mediated deubiquitination. a , b HEK293T cells were transfected with plasmids encoding Flag-caspase-1, Vsv-ASC, His-pro-IL-1β (WT) or His-pro-IL-1β-K133R with different amounts (125, 250, 500 ng) of plasmid encoding Flag-POH1 and 500 ng of control plasmid, the secretion of a IL-1β in the supernatants was quantified by ELISA 36 h after transfection; b the cell lysates were IB with the indicated antibodies. c Mutants of murine pro-IL-1β with replacement of various lysine residues. d-f HEK293T cells were transfected with Flag-POH1, V5-caspase-1, V5-ASC, His-pro-IL-1β (WT) or its mutants, along d with or e-f without HA-tagged K63-only Ub, then d cells were subjected to IP with an anti-His antibody and IB with the indicated antibodies. e The IL-1β levels in supernatants were measured by ELISA; or f the cell lysates were collected and IB with the indicated antibodies. g Proposed function of POH1 as a negative regulator in mediating IL-1β processing. POH1, upregulated by TLR3/4 activation, interacts with and deubiquitinates pro-IL-1β, resulting in restriction of IL-1β cleavage and secretion. Similar results were obtained from three independent experiments. The results represent the mean ± s.d. of three independent sets of experiments. ** p
    Figure Legend Snippet: Pro-IL-1β maturation is regulated by POH1-mediated deubiquitination. a , b HEK293T cells were transfected with plasmids encoding Flag-caspase-1, Vsv-ASC, His-pro-IL-1β (WT) or His-pro-IL-1β-K133R with different amounts (125, 250, 500 ng) of plasmid encoding Flag-POH1 and 500 ng of control plasmid, the secretion of a IL-1β in the supernatants was quantified by ELISA 36 h after transfection; b the cell lysates were IB with the indicated antibodies. c Mutants of murine pro-IL-1β with replacement of various lysine residues. d-f HEK293T cells were transfected with Flag-POH1, V5-caspase-1, V5-ASC, His-pro-IL-1β (WT) or its mutants, along d with or e-f without HA-tagged K63-only Ub, then d cells were subjected to IP with an anti-His antibody and IB with the indicated antibodies. e The IL-1β levels in supernatants were measured by ELISA; or f the cell lysates were collected and IB with the indicated antibodies. g Proposed function of POH1 as a negative regulator in mediating IL-1β processing. POH1, upregulated by TLR3/4 activation, interacts with and deubiquitinates pro-IL-1β, resulting in restriction of IL-1β cleavage and secretion. Similar results were obtained from three independent experiments. The results represent the mean ± s.d. of three independent sets of experiments. ** p

    Techniques Used: Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Activation Assay

    POH1 expression is upregulated in macrophages by TLR3/4 activation. a BMDMs from WT mice were stimulated with LPS (100 ng/mL), poly(I:C) (10 μg/mL), CpG (5 μM) or IL-1β (10 ng/mL) for 9 h and analysed for expression of POH1 by immunoblot. b, c Immunoblot analysis of POH1 expression from BMDMs treated with b LPS or ( c ) poly(I:C) as indicated. d, e RT-PCR analysis of POH1 expression in BMDMs in response to d LPS or e poly(I:C) stimulation. Similar results were obtained from three independent experiments. The results represent the mean ± s.d. of three independent sets of experiments
    Figure Legend Snippet: POH1 expression is upregulated in macrophages by TLR3/4 activation. a BMDMs from WT mice were stimulated with LPS (100 ng/mL), poly(I:C) (10 μg/mL), CpG (5 μM) or IL-1β (10 ng/mL) for 9 h and analysed for expression of POH1 by immunoblot. b, c Immunoblot analysis of POH1 expression from BMDMs treated with b LPS or ( c ) poly(I:C) as indicated. d, e RT-PCR analysis of POH1 expression in BMDMs in response to d LPS or e poly(I:C) stimulation. Similar results were obtained from three independent experiments. The results represent the mean ± s.d. of three independent sets of experiments

    Techniques Used: Expressing, Activation Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

    Effects of POH1 expression on inflammasome-mediated ASC foci formation. a-c Poh1 Δ/+ and Poh1 Δ/Δ BMDMs were primed with LPS for 12–14 h and then stimulated with ATP (0.5 h), nigericin (Nig, 0.5 h), poly(dA:dT) (5 h) or flagellin (5 h) as indicated, a cells were then fixed and stained for ASC (green) and DNA (blue) (scale bars, 10 µm); b percentages of macrophages containing ASC foci were calculated; or c cells harvested after different treatments were analysed for ASC polymerization. d , e Immunofluorescence microscopy of BMDMs transduced with control (Vector) or POH1, the cells were treated as in a , d and then stained for ASC (green) and DNA (blue) (scale bars, 10 µm); e percentages of macrophages containing ASC foci. f Immunoblot analysis of ASC polymerization in BMDMs treated as in d . g , h HEK293T cells were transfected with 200 ng of plasmids encoding Flag-pro-caspase-1, Flag-ASC and His-pro-IL-1β with different amounts (31.25, 62.5, 125, 250, 500 ng) of plasmid encoding Flag-POH1 and 500 ng of control plasmid, g the cell lysates were immunoblotted with the indicated antibodies; h IL-1β levels in supernatants were analysed by ELISA 36 h after transfection. Similar results were obtained from three independent experiments. The results represent the mean ± s.d. of three independent sets of experiments. *** p
    Figure Legend Snippet: Effects of POH1 expression on inflammasome-mediated ASC foci formation. a-c Poh1 Δ/+ and Poh1 Δ/Δ BMDMs were primed with LPS for 12–14 h and then stimulated with ATP (0.5 h), nigericin (Nig, 0.5 h), poly(dA:dT) (5 h) or flagellin (5 h) as indicated, a cells were then fixed and stained for ASC (green) and DNA (blue) (scale bars, 10 µm); b percentages of macrophages containing ASC foci were calculated; or c cells harvested after different treatments were analysed for ASC polymerization. d , e Immunofluorescence microscopy of BMDMs transduced with control (Vector) or POH1, the cells were treated as in a , d and then stained for ASC (green) and DNA (blue) (scale bars, 10 µm); e percentages of macrophages containing ASC foci. f Immunoblot analysis of ASC polymerization in BMDMs treated as in d . g , h HEK293T cells were transfected with 200 ng of plasmids encoding Flag-pro-caspase-1, Flag-ASC and His-pro-IL-1β with different amounts (31.25, 62.5, 125, 250, 500 ng) of plasmid encoding Flag-POH1 and 500 ng of control plasmid, g the cell lysates were immunoblotted with the indicated antibodies; h IL-1β levels in supernatants were analysed by ELISA 36 h after transfection. Similar results were obtained from three independent experiments. The results represent the mean ± s.d. of three independent sets of experiments. *** p

    Techniques Used: Expressing, Staining, Immunofluorescence, Microscopy, Transduction, Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay

    POH1 inhibits inflammasome-induced pro-IL-1β processing. a-c BMDMs from Poh1 Δ/+ and Poh1 Δ/Δ mice were primed with LPS for 12–14 h and then stimulated with ATP (0.5 h), nigericin (Nig, 0.5 h), poly(dA:dT) (5 h) or flagellin (5 h) as indicated, a IL-1β, b IL-6 and c TNFα levels in supernatants were analysed by ELISA. d-f BMDMs lentivirally transduced with control (Vector) or POH1 were stimulated as in a , d IL-1β, e IL-6 and f TNFα levels in supernatants were analysed by ELISA. g, h BMDMs g derived from Poh1 Δ/+ and Poh1 Δ/Δ mice or h infected by indicated lentiviruses were treated as in a , the cell lysates (CL) and supernatants (SN) were collected and immunoblotted with the indicated antibodies. Similar results were obtained from three independent experiments. The results represent the mean ± s.d. of three independent sets of experiments. ** p
    Figure Legend Snippet: POH1 inhibits inflammasome-induced pro-IL-1β processing. a-c BMDMs from Poh1 Δ/+ and Poh1 Δ/Δ mice were primed with LPS for 12–14 h and then stimulated with ATP (0.5 h), nigericin (Nig, 0.5 h), poly(dA:dT) (5 h) or flagellin (5 h) as indicated, a IL-1β, b IL-6 and c TNFα levels in supernatants were analysed by ELISA. d-f BMDMs lentivirally transduced with control (Vector) or POH1 were stimulated as in a , d IL-1β, e IL-6 and f TNFα levels in supernatants were analysed by ELISA. g, h BMDMs g derived from Poh1 Δ/+ and Poh1 Δ/Δ mice or h infected by indicated lentiviruses were treated as in a , the cell lysates (CL) and supernatants (SN) were collected and immunoblotted with the indicated antibodies. Similar results were obtained from three independent experiments. The results represent the mean ± s.d. of three independent sets of experiments. ** p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Transduction, Plasmid Preparation, Derivative Assay, Infection

    22) Product Images from "Treatment with catalpol protects against cisplatin-induced renal injury through Nrf2 and NF-κB signaling pathways"

    Article Title: Treatment with catalpol protects against cisplatin-induced renal injury through Nrf2 and NF-κB signaling pathways

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2020.9077

    Effects of catalpol on the CP-stimulated production of inflammatory cytokines in the kidney tissues. Expression levels of (A) TNF-α, (B) IL-1β, (C) IL-6, (D) IL-8, (E) IL-10 and (F) iNOS in the kidney tissues of different treatment groups were determined using ELISAs. Results are presented as the mean ± SD (n=8). # P
    Figure Legend Snippet: Effects of catalpol on the CP-stimulated production of inflammatory cytokines in the kidney tissues. Expression levels of (A) TNF-α, (B) IL-1β, (C) IL-6, (D) IL-8, (E) IL-10 and (F) iNOS in the kidney tissues of different treatment groups were determined using ELISAs. Results are presented as the mean ± SD (n=8). # P

    Techniques Used: Expressing

    23) Product Images from "Polyphenols Derived from Lychee Seed Suppress Aβ (1-42)-Induced Neuroinflammation"

    Article Title: Polyphenols Derived from Lychee Seed Suppress Aβ (1-42)-Induced Neuroinflammation

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19072109

    The release of TNF-α, IL-1β, and IL-6 in Aβ(1-42)-induced BV-2 cells under the treatments of LSF, catechin, and procyanidin A2. BV-2 cells were pretreated with 5 µM Aβ(1-42) for 12 h, followed by incubations of LSF (0.469 µg/mL), catechin (10 µM), and procyanidin A2 (10 µM) for another 12 h. After treatment, the cell free supernatants were subsequently employed for TNF-α ( A ), IL-1β ( B ), and IL-6 ( C ) assays using ELISA kit. BV-2 cells treated with medium alone were set as the control group, and BV-2 cells treated with 5 µM Aβ(1-42) alone were set as the Aβ group. ### p
    Figure Legend Snippet: The release of TNF-α, IL-1β, and IL-6 in Aβ(1-42)-induced BV-2 cells under the treatments of LSF, catechin, and procyanidin A2. BV-2 cells were pretreated with 5 µM Aβ(1-42) for 12 h, followed by incubations of LSF (0.469 µg/mL), catechin (10 µM), and procyanidin A2 (10 µM) for another 12 h. After treatment, the cell free supernatants were subsequently employed for TNF-α ( A ), IL-1β ( B ), and IL-6 ( C ) assays using ELISA kit. BV-2 cells treated with medium alone were set as the control group, and BV-2 cells treated with 5 µM Aβ(1-42) alone were set as the Aβ group. ### p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    The mRNA levels of TNF-α, IL-1β, and iNOS in Aβ(1-42)-induced BV-2 cells under the treatments of LSF, catechin, and procyanidin A2. BV-2 cells were pretreated with 5 µM Aβ(1-42) for 12 h, followed by incubations of LSF (0.469 µg/mL), catechin (10 µM), and procyanidin A2 (10 µM) for another 12 h. After treatment, total mRNA were extracted and subsequently employed for TNF-α ( A ), IL-1β ( B ), and iNOS ( C ) measurements using RT-PCR. BV-2 cells treated with medium alone were set as the control group, and BV-2 cells treated with 5 µM Aβ(1-42) alone were set as the Aβ(1-42) group. ### p
    Figure Legend Snippet: The mRNA levels of TNF-α, IL-1β, and iNOS in Aβ(1-42)-induced BV-2 cells under the treatments of LSF, catechin, and procyanidin A2. BV-2 cells were pretreated with 5 µM Aβ(1-42) for 12 h, followed by incubations of LSF (0.469 µg/mL), catechin (10 µM), and procyanidin A2 (10 µM) for another 12 h. After treatment, total mRNA were extracted and subsequently employed for TNF-α ( A ), IL-1β ( B ), and iNOS ( C ) measurements using RT-PCR. BV-2 cells treated with medium alone were set as the control group, and BV-2 cells treated with 5 µM Aβ(1-42) alone were set as the Aβ(1-42) group. ### p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    The protein expressions of TNF-α, IL-1β, and iNOS in Aβ(1-42)-induced BV-2 cells under the treatments of LSF (0.469 µg/mL), catechin (10 µM), and procyanidin A2 (10 µM). BV-2 cells were pretreated with 5 µM Aβ(1-42) for 12 h, followed by incubations of LSF, catechin, and procyanidin A2 for another 12 h. ( A ) After treatment, cell lysates were harvested and subsequently employed for TNF-α, IL-1β, and iNOS measurements using Western blotting. Band intensities of TNF-α ( B ), IL-1β ( C ), and iNOS ( D ) were quantified using Image J software and normalized to β-actin. Bars are representatives of three independent experiments. BV-2 cells treated with medium alone were set as the control group, and BV-2 cells treated with 5 µM Aβ(1-42) alone were set as the Aβ group. # p
    Figure Legend Snippet: The protein expressions of TNF-α, IL-1β, and iNOS in Aβ(1-42)-induced BV-2 cells under the treatments of LSF (0.469 µg/mL), catechin (10 µM), and procyanidin A2 (10 µM). BV-2 cells were pretreated with 5 µM Aβ(1-42) for 12 h, followed by incubations of LSF, catechin, and procyanidin A2 for another 12 h. ( A ) After treatment, cell lysates were harvested and subsequently employed for TNF-α, IL-1β, and iNOS measurements using Western blotting. Band intensities of TNF-α ( B ), IL-1β ( C ), and iNOS ( D ) were quantified using Image J software and normalized to β-actin. Bars are representatives of three independent experiments. BV-2 cells treated with medium alone were set as the control group, and BV-2 cells treated with 5 µM Aβ(1-42) alone were set as the Aβ group. # p

    Techniques Used: Western Blot, Software

    24) Product Images from "Targeting connexin 43 provides anti-inflammatory effects after intracerebral hemorrhage injury by regulating YAP signaling"

    Article Title: Targeting connexin 43 provides anti-inflammatory effects after intracerebral hemorrhage injury by regulating YAP signaling

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-020-01978-z

    In vitro, the YAP inhibitor VP reverses the regulatory effect of Gap19 on the Cx43-YAP-SOCS axis. a , b At the beginning of hemin stimulation, astrocytes were treated with the YAP inhibitor VP. WB analysis showed that VP suppressed the increase in SOCS1/SOCS3 levels induced by Gap19 treatment. c , d Representative photos showing the levels of TLR4, p-IKKβ, IKKβ, IKBα, p65, p-p65, and β-tubulin after treatment with the YAP inhibitor VP. e , f Representative photographs showing the levels of JAK2, p-JAK2, STAT3, p-STAT3, and β-tubulin after treatment with the YAP inhibitor VP. g qRT-PCR analysis of the transcriptional activities of IL-1β, IL-6, TNF-α, and MCP1 in astrocytes after VP treatment. The bars represent the SEM of the data from 3 samples per group. * P
    Figure Legend Snippet: In vitro, the YAP inhibitor VP reverses the regulatory effect of Gap19 on the Cx43-YAP-SOCS axis. a , b At the beginning of hemin stimulation, astrocytes were treated with the YAP inhibitor VP. WB analysis showed that VP suppressed the increase in SOCS1/SOCS3 levels induced by Gap19 treatment. c , d Representative photos showing the levels of TLR4, p-IKKβ, IKKβ, IKBα, p65, p-p65, and β-tubulin after treatment with the YAP inhibitor VP. e , f Representative photographs showing the levels of JAK2, p-JAK2, STAT3, p-STAT3, and β-tubulin after treatment with the YAP inhibitor VP. g qRT-PCR analysis of the transcriptional activities of IL-1β, IL-6, TNF-α, and MCP1 in astrocytes after VP treatment. The bars represent the SEM of the data from 3 samples per group. * P

    Techniques Used: In Vitro, Western Blot, Quantitative RT-PCR

    Gap19 reduces the production and release of proinflammatory cytokines by astrocytes after ICH injury. a qRT-PCR analysis of the transcriptional activities of IL-1β, IL-6, TNF-α, and MCP-1 in astrocytes. b – e ELISA was used to detect the release of IL-1β, IL-6, TNF-α, and MCP1 from astrocytes to the supernatant following different treatments. The bars represent the SEM of data from 3 samples per group. * P
    Figure Legend Snippet: Gap19 reduces the production and release of proinflammatory cytokines by astrocytes after ICH injury. a qRT-PCR analysis of the transcriptional activities of IL-1β, IL-6, TNF-α, and MCP-1 in astrocytes. b – e ELISA was used to detect the release of IL-1β, IL-6, TNF-α, and MCP1 from astrocytes to the supernatant following different treatments. The bars represent the SEM of data from 3 samples per group. * P

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

    Gap19 reduces the expression of proinflammatory cytokines and the activation of astrocytes after ICH injury. a , b WB analysis showed the expression levels of TNF-α, IL-1β, IL-6, MCP1, and β-tubulin as the loading control. c , d The expression levels of IL-4 and IL-10. β-Tubulin was used as a loading control. e , f The EtBr uptake assay was used to evaluate the activity of Hcs on astrocytes, microglia, and neurons after hemin stimulation. Gap19 inhibits the hemin-induced excessive opening of Hcs on astrocytes. g , h Immunofluorescence for GFAP was used to evaluate astrocyte activation. TAT-Gap19 treatment significantly reduced the number of GFAP-positive cells in the area surrounding cerebral hemorrhage. i , j In vitro, Gap19 treatment reduced the GFAP fluorescence intensity in astrocytes after hemin stimulation. The bars in a – d , g , and h represent the SEM of the data from 3 cerebral hemorrhage tissue samples per group. * P
    Figure Legend Snippet: Gap19 reduces the expression of proinflammatory cytokines and the activation of astrocytes after ICH injury. a , b WB analysis showed the expression levels of TNF-α, IL-1β, IL-6, MCP1, and β-tubulin as the loading control. c , d The expression levels of IL-4 and IL-10. β-Tubulin was used as a loading control. e , f The EtBr uptake assay was used to evaluate the activity of Hcs on astrocytes, microglia, and neurons after hemin stimulation. Gap19 inhibits the hemin-induced excessive opening of Hcs on astrocytes. g , h Immunofluorescence for GFAP was used to evaluate astrocyte activation. TAT-Gap19 treatment significantly reduced the number of GFAP-positive cells in the area surrounding cerebral hemorrhage. i , j In vitro, Gap19 treatment reduced the GFAP fluorescence intensity in astrocytes after hemin stimulation. The bars in a – d , g , and h represent the SEM of the data from 3 cerebral hemorrhage tissue samples per group. * P

    Techniques Used: Expressing, Activation Assay, Western Blot, Activity Assay, Immunofluorescence, In Vitro, Fluorescence

    25) Product Images from "Trimethylamine‐N‐Oxide Induces Vascular Inflammation by Activating the NLRP3 Inflammasome Through the SIRT3‐SOD2‐mtROS Signaling Pathway"

    Article Title: Trimethylamine‐N‐Oxide Induces Vascular Inflammation by Activating the NLRP3 Inflammasome Through the SIRT3‐SOD2‐mtROS Signaling Pathway

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    doi: 10.1161/JAHA.117.006347

    Trimethylamine‐N‐oxide ( TMAO ) induced mitochondrial reactive oxygen species (mt ROS ) accumulation via the sirtuin‐3–superoxide dismutase‐2 ( SIRT 3‐ SOD 2) pathway in endothelial cells. Human umbilical vein endothelial cells ( HUVEC s) were transfected with SIRT 3 si RNA or a plasmid overexpressing SIRT 3 as described in Materials and Methods . After 24 hours, cells were incubated with 600 μmol/L TMAO for 24 hours. A, Assay of SOD 2 enzymatic activity using SOD 1 and SOD 2 Assay Kits with WST ‐8 following the manufacturer's instructions. B, Detection of mt ROS levels with Mito SOX ™ Red. C, Detection of total ROS levels using DCFH ‐ DA . D, Measurement of caspase‐1 activity with a caspase‐1 activity assay kit. E, Western blot analysis of caspase‐1 p20, nucleotide‐binding oligomerization domain–like receptor family pyrin domain–containing 3 ( NLRP 3), Ac‐ SOD 2, SOD 2, and SIRT 3 contents. F, Bar graphs showing quantification of the indicated proteins. G, Western blot analysis of IL ‐1β, ICAM ‐1, and MMP ‐9 expression. H, Bar graphs showing quantification of the indicated proteins. Values are expressed as means± SE (n=3); b P
    Figure Legend Snippet: Trimethylamine‐N‐oxide ( TMAO ) induced mitochondrial reactive oxygen species (mt ROS ) accumulation via the sirtuin‐3–superoxide dismutase‐2 ( SIRT 3‐ SOD 2) pathway in endothelial cells. Human umbilical vein endothelial cells ( HUVEC s) were transfected with SIRT 3 si RNA or a plasmid overexpressing SIRT 3 as described in Materials and Methods . After 24 hours, cells were incubated with 600 μmol/L TMAO for 24 hours. A, Assay of SOD 2 enzymatic activity using SOD 1 and SOD 2 Assay Kits with WST ‐8 following the manufacturer's instructions. B, Detection of mt ROS levels with Mito SOX ™ Red. C, Detection of total ROS levels using DCFH ‐ DA . D, Measurement of caspase‐1 activity with a caspase‐1 activity assay kit. E, Western blot analysis of caspase‐1 p20, nucleotide‐binding oligomerization domain–like receptor family pyrin domain–containing 3 ( NLRP 3), Ac‐ SOD 2, SOD 2, and SIRT 3 contents. F, Bar graphs showing quantification of the indicated proteins. G, Western blot analysis of IL ‐1β, ICAM ‐1, and MMP ‐9 expression. H, Bar graphs showing quantification of the indicated proteins. Values are expressed as means± SE (n=3); b P

    Techniques Used: Transfection, Plasmid Preparation, Incubation, Activity Assay, Western Blot, Binding Assay, Expressing

    Trimethylamine‐N‐oxide ( TMAO )‐induced inflammation in endothelial cells. A, Human umbilical vein endothelial cells ( HUVEC s) were incubated with different concentrations of TMAO (50, 100, 200, 300, 400, 600, 800, 1000, and 2000 μmol/L) for 24 hours. Thereafter, cell viability was determined. B, Cells were treated with TMAO (600 μmol/L) for different time‐periods (4, 8, 12, 24, 36, 48, and 72 hours), and cell viability was detected. C, Cells were treated as described in A, and the expression of IL ‐1β, ICAM ‐1, and MMP ‐9 was detected via Western blot. D, Bar charts show the quantification of the indicated proteins. E, Cells were treated as described in B, and the expression of IL ‐1β, ICAM ‐1, and MMP ‐9 was analyzed via Western blot. F, Bar graphs show the quantification of the indicated proteins. Values are presented as means± SE (n=3); a P
    Figure Legend Snippet: Trimethylamine‐N‐oxide ( TMAO )‐induced inflammation in endothelial cells. A, Human umbilical vein endothelial cells ( HUVEC s) were incubated with different concentrations of TMAO (50, 100, 200, 300, 400, 600, 800, 1000, and 2000 μmol/L) for 24 hours. Thereafter, cell viability was determined. B, Cells were treated with TMAO (600 μmol/L) for different time‐periods (4, 8, 12, 24, 36, 48, and 72 hours), and cell viability was detected. C, Cells were treated as described in A, and the expression of IL ‐1β, ICAM ‐1, and MMP ‐9 was detected via Western blot. D, Bar charts show the quantification of the indicated proteins. E, Cells were treated as described in B, and the expression of IL ‐1β, ICAM ‐1, and MMP ‐9 was analyzed via Western blot. F, Bar graphs show the quantification of the indicated proteins. Values are presented as means± SE (n=3); a P

    Techniques Used: Incubation, Expressing, Western Blot

    Trimethylamine‐N‐oxide ( TMAO )‐induced inflammation via nucleotide‐binding oligomerization domain–like receptor family pyrin domain–containing 3 ( NLRP 3) inflammasome activation in endothelial cells. A, Cells were treated with TMAO at a series of concentrations (150, 300, 600, and 900 μmol/L) for 24 hours, and the expression of NLRP 3 and caspase‐1 p20 was detected via Western blot. B, Bar charts showing quantification of endogenous NLRP 3 and caspase‐1 p20. C, Cells were incubated with 600 μmol/L TMAO for different time intervals (4, 8, 12, and 24 hours), and the expression of NLRP 3 and caspase‐1 p20 was detected via Western blot. D, Bar charts showing quantification of endogenous NLRP 3 and caspase‐1 p20. Cells were treated as described for A and B. Thereafter, caspase‐1 activity was measured using caspase‐1 activity kits (E and F). G, Cells were pretreated with YVAD (10 μmol/L) or MCC 950 (10 μmol/L) for 2 hours, and then exposed to TMAO (600 μmol/L) for a further 24 hours. Expression of IL ‐1β, ICAM ‐1, MMP ‐9, and caspase‐1 p20 was detected via Western blot. H, Bar charts showing quantification of the indicated proteins. I, Human umbilical vein endothelial cells ( HUVEC s) were transfected with NLRP 3 si RNA as described in Materials and Methods . After 24 hours, cells were incubated with TMAO (600 μmol/L) for 24 hours, and the expression of IL ‐1β, ICAM ‐1, MMP ‐9 and Casp1 p20 was detected via Western blot. J, Bar charts showing quantification of indicated proteins. Values are expressed as means± SE (n=3). a P
    Figure Legend Snippet: Trimethylamine‐N‐oxide ( TMAO )‐induced inflammation via nucleotide‐binding oligomerization domain–like receptor family pyrin domain–containing 3 ( NLRP 3) inflammasome activation in endothelial cells. A, Cells were treated with TMAO at a series of concentrations (150, 300, 600, and 900 μmol/L) for 24 hours, and the expression of NLRP 3 and caspase‐1 p20 was detected via Western blot. B, Bar charts showing quantification of endogenous NLRP 3 and caspase‐1 p20. C, Cells were incubated with 600 μmol/L TMAO for different time intervals (4, 8, 12, and 24 hours), and the expression of NLRP 3 and caspase‐1 p20 was detected via Western blot. D, Bar charts showing quantification of endogenous NLRP 3 and caspase‐1 p20. Cells were treated as described for A and B. Thereafter, caspase‐1 activity was measured using caspase‐1 activity kits (E and F). G, Cells were pretreated with YVAD (10 μmol/L) or MCC 950 (10 μmol/L) for 2 hours, and then exposed to TMAO (600 μmol/L) for a further 24 hours. Expression of IL ‐1β, ICAM ‐1, MMP ‐9, and caspase‐1 p20 was detected via Western blot. H, Bar charts showing quantification of the indicated proteins. I, Human umbilical vein endothelial cells ( HUVEC s) were transfected with NLRP 3 si RNA as described in Materials and Methods . After 24 hours, cells were incubated with TMAO (600 μmol/L) for 24 hours, and the expression of IL ‐1β, ICAM ‐1, MMP ‐9 and Casp1 p20 was detected via Western blot. J, Bar charts showing quantification of indicated proteins. Values are expressed as means± SE (n=3). a P

    Techniques Used: Binding Assay, Activation Assay, Expressing, Western Blot, Incubation, Activity Assay, Transfection

    Trimethylamine‐N‐oxide ( TMAO ) induced vascular inflammation via nucleotide‐binding oligomerization domain–like receptor family pyrin domain–containing 3 ( NLRP 3) inflammasome activation in vivo. Eight‐week‐old female ApoE −/− mice (n=10 per group) were fed chow diet or chow diet combined with choline (1%) for 4 months. Mice were killed, and their blood and aorta samples were collected immediately, snap‐frozen in liquid nitrogen, and stored at −80°C until required. A, Ultrasound B‐mode images of aortic sinus and quantification. Arrows indicate the regions of interest. B, Oil‐red O staining of whole aortas, including aortic arch and thoracic and abdominal regions, and their quantitation. C, Oil‐red O–stained aortic root (counterstained with hematoxylin) and quantification. D, Western blot analysis of caspase‐1 p20, NLRP 3, IL ‐1β, ICAM ‐1, and MMP ‐9 contents in aortas. E, Bar graphs showing quantification of the indicated proteins. F, Caspase‐1 activity in aortas. G, Measurement of plasma TMAO levels using liquid chromatography–tandem mass spectrometry. Values are expressed as means± SE (n=10). * P
    Figure Legend Snippet: Trimethylamine‐N‐oxide ( TMAO ) induced vascular inflammation via nucleotide‐binding oligomerization domain–like receptor family pyrin domain–containing 3 ( NLRP 3) inflammasome activation in vivo. Eight‐week‐old female ApoE −/− mice (n=10 per group) were fed chow diet or chow diet combined with choline (1%) for 4 months. Mice were killed, and their blood and aorta samples were collected immediately, snap‐frozen in liquid nitrogen, and stored at −80°C until required. A, Ultrasound B‐mode images of aortic sinus and quantification. Arrows indicate the regions of interest. B, Oil‐red O staining of whole aortas, including aortic arch and thoracic and abdominal regions, and their quantitation. C, Oil‐red O–stained aortic root (counterstained with hematoxylin) and quantification. D, Western blot analysis of caspase‐1 p20, NLRP 3, IL ‐1β, ICAM ‐1, and MMP ‐9 contents in aortas. E, Bar graphs showing quantification of the indicated proteins. F, Caspase‐1 activity in aortas. G, Measurement of plasma TMAO levels using liquid chromatography–tandem mass spectrometry. Values are expressed as means± SE (n=10). * P

    Techniques Used: Binding Assay, Activation Assay, In Vivo, Mouse Assay, Staining, Quantitation Assay, Western Blot, Activity Assay, Liquid Chromatography, Mass Spectrometry

    Trimethylamine‐N‐oxide ( TMAO ) induced vascular nucleotide‐binding oligomerization domain–like receptor family pyrin domain–containing 3 ( NLRP 3) inflammasome activation in a sirtuin‐3 ( SIRT 3)‐dependent manner in vivo. Eight‐week‐old female ApoE −/− mice were treated with or without 1% choline for 4 months. Mice were killed, and their aorta samples were collected immediately, snap‐frozen in liquid nitrogen, and stored at −80°C until required. A, Western blot detection of Ac‐ SOD 2, SOD 2, and SIRT 3 expression in aortas. B, Bar graphs showing quantification of the indicated proteins. Eight‐week‐old female wild type ( WT ) and SIRT 3 −/− mice were fed with or without 1% choline for 4 months. Mice were killed, and their aorta samples were collected immediately, snap‐frozen in liquid nitrogen, and stored at −80°C until required. C, Western blot analysis of caspase‐1 p20, NLRP 3, Ac‐ SOD 2, SOD 2, and SIRT 3 contents in aortas. D, Bar graphs showing quantification of the indicated proteins. E, SOD 2 enzymatic activity in aortas was assayed using a SOD 1 and SOD 2 Assay Kit with WST ‐8 following the manufacturer's instructions. F, Measurement of caspase‐1 activity in aortas. G, Western blot analysis of IL ‐1β, ICAM ‐1, MMP ‐9 and SIRT 3 expression. H, Bar graphs showing quantification of the indicated proteins. Values are expressed as means± SE (n=10); * P
    Figure Legend Snippet: Trimethylamine‐N‐oxide ( TMAO ) induced vascular nucleotide‐binding oligomerization domain–like receptor family pyrin domain–containing 3 ( NLRP 3) inflammasome activation in a sirtuin‐3 ( SIRT 3)‐dependent manner in vivo. Eight‐week‐old female ApoE −/− mice were treated with or without 1% choline for 4 months. Mice were killed, and their aorta samples were collected immediately, snap‐frozen in liquid nitrogen, and stored at −80°C until required. A, Western blot detection of Ac‐ SOD 2, SOD 2, and SIRT 3 expression in aortas. B, Bar graphs showing quantification of the indicated proteins. Eight‐week‐old female wild type ( WT ) and SIRT 3 −/− mice were fed with or without 1% choline for 4 months. Mice were killed, and their aorta samples were collected immediately, snap‐frozen in liquid nitrogen, and stored at −80°C until required. C, Western blot analysis of caspase‐1 p20, NLRP 3, Ac‐ SOD 2, SOD 2, and SIRT 3 contents in aortas. D, Bar graphs showing quantification of the indicated proteins. E, SOD 2 enzymatic activity in aortas was assayed using a SOD 1 and SOD 2 Assay Kit with WST ‐8 following the manufacturer's instructions. F, Measurement of caspase‐1 activity in aortas. G, Western blot analysis of IL ‐1β, ICAM ‐1, MMP ‐9 and SIRT 3 expression. H, Bar graphs showing quantification of the indicated proteins. Values are expressed as means± SE (n=10); * P

    Techniques Used: Binding Assay, Activation Assay, In Vivo, Mouse Assay, Western Blot, Expressing, Activity Assay

    26) Product Images from "Restoration of the type I IFN–IL-1 balance through targeted blockade of PTGER4 inhibits autoimmunity in NOD mice"

    Article Title: Restoration of the type I IFN–IL-1 balance through targeted blockade of PTGER4 inhibits autoimmunity in NOD mice

    Journal: JCI Insight

    doi: 10.1172/jci.insight.97843

    PGE2-PTGER4–driven signals are inflammatory in NOD mice. ( A and B ) Spleen cells from 8-week-old NOD mice were stimulated with LPS-ATP. After 4 hours of LPS stimulation, where indicated PGE2 was added to the culture for 30 minutes, followed by ATP stimulation for 1 hour. Cells lysates ( A ) or supernatant ( B ) were analyzed by Western blotting for IL-1β and NLRP3. Protein bands were quantified relative to LAMIN-B ( A ) or total cell number ( B ). ( C ) CD11c + cells were enriched and stimulated in vitro with IFN-α or LPS or both and PTGER4 (EP4) antagonist (EP4BL) as indicated. Transcripts of IL-1β and Nlrp3 were measured 2 hours after stimulation. ( D ) Total spleen cells were stimulated with or without LPS with or without PTGER4 antagonist. Intracellular IFN-γ was measured 6 hours after stimulation in CD4 + T cells. Data are representative of at least 2 ( A and B ) or 3 ( C and D ) independent experiments performed with pooled mice ( n = 3), mean ± SD. Statistical analysis was performed with 1-way ANOVA with Bonferroni post-test, and only the relevant comparison was depicted ( C and D ). ** P
    Figure Legend Snippet: PGE2-PTGER4–driven signals are inflammatory in NOD mice. ( A and B ) Spleen cells from 8-week-old NOD mice were stimulated with LPS-ATP. After 4 hours of LPS stimulation, where indicated PGE2 was added to the culture for 30 minutes, followed by ATP stimulation for 1 hour. Cells lysates ( A ) or supernatant ( B ) were analyzed by Western blotting for IL-1β and NLRP3. Protein bands were quantified relative to LAMIN-B ( A ) or total cell number ( B ). ( C ) CD11c + cells were enriched and stimulated in vitro with IFN-α or LPS or both and PTGER4 (EP4) antagonist (EP4BL) as indicated. Transcripts of IL-1β and Nlrp3 were measured 2 hours after stimulation. ( D ) Total spleen cells were stimulated with or without LPS with or without PTGER4 antagonist. Intracellular IFN-γ was measured 6 hours after stimulation in CD4 + T cells. Data are representative of at least 2 ( A and B ) or 3 ( C and D ) independent experiments performed with pooled mice ( n = 3), mean ± SD. Statistical analysis was performed with 1-way ANOVA with Bonferroni post-test, and only the relevant comparison was depicted ( C and D ). ** P

    Techniques Used: Mouse Assay, Western Blot, In Vitro

    Inflammatory signature identified in NOD APCs correlates with increased PTGER4 and PGE2. ( A ) Z scores for IPA signaling pathways significantly altered in moDCs and cDC2 from NOD versus B6.g7 mice. ( B ) Bubble plots of key inflammatory gene sets from GSEA of NOD APCs compared with B6.g7 APCs using the immunologic signatures gene sets from MSigDB. Gene sets enriched with genes with lower expression in NOD mice compared with B6.g7 mice are depicted as blue circles, while gene sets with higher expression in NOD mice are depicted as red circles. Circle size indicates FDR-adjusted P values, and color intensity indicates normalized enrichment scores. ( C ) Hierarchical clustering of significant IPA upstream regulators with increased expression in NOD mice as ranked by Z score, which indicates the predicted enrichment in NOD mice compared with B6.g7 mice. ( D ) Gene expression measured by qPCR for IL-1β, Nlrp3, and Ptger4 after 4 hours of LPS stimulation of spleen cells from 8-week-old prediabetic NOD and B6.g7 mice. Plots are representative of 2 independent experiments with 3 mice per group. ( E ) PGE2 levels in sera from NOD and B6.g7 mice (representative of 2 experiments, 3 mice per group) at weeks 3 and 8. Comparison was made between NOD and B6.g7 mice; P values were calculated using t test. Data show mean ± SD. ( F and G ) Protein expression level by Western blot of PTGER4 and PTGER2 in bone marrow–derived DCs from NOD and B6.g7 mice, from 0–4 hours after LPS (500 ng/ml) stimulation. Relative quantification of bands normalization to HSP90 is indicated underneath. *P
    Figure Legend Snippet: Inflammatory signature identified in NOD APCs correlates with increased PTGER4 and PGE2. ( A ) Z scores for IPA signaling pathways significantly altered in moDCs and cDC2 from NOD versus B6.g7 mice. ( B ) Bubble plots of key inflammatory gene sets from GSEA of NOD APCs compared with B6.g7 APCs using the immunologic signatures gene sets from MSigDB. Gene sets enriched with genes with lower expression in NOD mice compared with B6.g7 mice are depicted as blue circles, while gene sets with higher expression in NOD mice are depicted as red circles. Circle size indicates FDR-adjusted P values, and color intensity indicates normalized enrichment scores. ( C ) Hierarchical clustering of significant IPA upstream regulators with increased expression in NOD mice as ranked by Z score, which indicates the predicted enrichment in NOD mice compared with B6.g7 mice. ( D ) Gene expression measured by qPCR for IL-1β, Nlrp3, and Ptger4 after 4 hours of LPS stimulation of spleen cells from 8-week-old prediabetic NOD and B6.g7 mice. Plots are representative of 2 independent experiments with 3 mice per group. ( E ) PGE2 levels in sera from NOD and B6.g7 mice (representative of 2 experiments, 3 mice per group) at weeks 3 and 8. Comparison was made between NOD and B6.g7 mice; P values were calculated using t test. Data show mean ± SD. ( F and G ) Protein expression level by Western blot of PTGER4 and PTGER2 in bone marrow–derived DCs from NOD and B6.g7 mice, from 0–4 hours after LPS (500 ng/ml) stimulation. Relative quantification of bands normalization to HSP90 is indicated underneath. *P

    Techniques Used: Indirect Immunoperoxidase Assay, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Derivative Assay

    27) Product Images from "Quiescent and Proliferative Fibroblasts Exhibit Differential p300 HAT Activation through Control of 5-Methoxytryptophan Production"

    Article Title: Quiescent and Proliferative Fibroblasts Exhibit Differential p300 HAT Activation through Control of 5-Methoxytryptophan Production

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088507

    p300 HAT activation is decreased in pFb vs. SF-Fb. A). PMA-induced p300 HAT activity in untransfected native cells. Upper panel shows p300 proteins analyzed by Western blotting and lower panel, p300 HAT activity. B). p300 HAT activity in p300-transfected fibroblasts. Upper panel shows p300 proteins in Fb transfected with full-length p300 vectors and lower panel PMA-induced p300 HAT activity in the p300 overexpressing cells. Nuclear histone H1 was used as a loading control. C) and D). IL-1β- and TNFα-induced p300 HAT activities in p300-transfected SF-Fb or pFb. Each error bar denotes mean ± SEM (n = 3).
    Figure Legend Snippet: p300 HAT activation is decreased in pFb vs. SF-Fb. A). PMA-induced p300 HAT activity in untransfected native cells. Upper panel shows p300 proteins analyzed by Western blotting and lower panel, p300 HAT activity. B). p300 HAT activity in p300-transfected fibroblasts. Upper panel shows p300 proteins in Fb transfected with full-length p300 vectors and lower panel PMA-induced p300 HAT activity in the p300 overexpressing cells. Nuclear histone H1 was used as a loading control. C) and D). IL-1β- and TNFα-induced p300 HAT activities in p300-transfected SF-Fb or pFb. Each error bar denotes mean ± SEM (n = 3).

    Techniques Used: HAT Assay, Activation Assay, Activity Assay, Western Blot, Transfection

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    Article Snippet: Antibodies against HIF-1α (14179s), IL-1β (12426s), mTOR (2972s), and p-mTOR (5536s) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Blocking Assay:

    Article Title: The role of aquaporin 4 in apoptosis after intracerebral hemorrhage
    Article Snippet: .. After the blocking, the blotting membranes were incubated with specific primary antibodies as follows: rabbit polyclonal against the active cleaved fragment (17 kDa) of caspase-3 (1:1000; Idun Pharmaceuticals, Los Angeles, CA, USA), rabbit polyclonal directed against the active cleaved fragment (20 kDa) of caspase-8 (1:1000, Santa Cruz Biotechnology), rabbit polyclonal directed against the cleaved fragment of caspase-9 (38 kDa) (1:1000, CST, Boston, MA, USA), rabbit polyclonal against Bcl-2, mouse monoclonal against Bax antibodies (1:100, Santa Cruz Biotechnology), rabbit polyclonal against IL-1β receptor and IL-1β phosphorylation receptor (1:1000, CST), and rabbit polyclonal against TNF-α receptor I and TNF-α receptor I phosphorylation (1:1000, CST) for 2 h at room temperature and then with biotinylated goat anti-rabbit IgG (1: 200, Vector Labs) for 1 h at room temperature. .. The proteins were visualized using the ABC reagent (Vectastain ABC Kit, Vector Labs) and the ECL detection kit (Amersham Pharmacia Biotech, Baie-D’Urfe, QC, Canada).

    Article Title: Walnut Protein Hydrolysates Play a Protective Role on Neurotoxicity Induced by d-Galactose and Aluminum Chloride in Mice
    Article Snippet: .. After being blocked with a normal goat serum blocking solution for 20 min, the slices were incubated with primary antibodies of rabbit anti-TNF-α and anti-IL-1β (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. .. After washing three times with PBS, the slices were incubated with goat anti-rabbit secondary antibody for 20 min at 37 °C, and subsequently reacted with horseradish peroxidase-streptavidin (Streptavidin Peroxidase Link Detection Kits; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) for 20 min at room temperature.

    Incubation:

    Article Title: The role of aquaporin 4 in apoptosis after intracerebral hemorrhage
    Article Snippet: .. After the blocking, the blotting membranes were incubated with specific primary antibodies as follows: rabbit polyclonal against the active cleaved fragment (17 kDa) of caspase-3 (1:1000; Idun Pharmaceuticals, Los Angeles, CA, USA), rabbit polyclonal directed against the active cleaved fragment (20 kDa) of caspase-8 (1:1000, Santa Cruz Biotechnology), rabbit polyclonal directed against the cleaved fragment of caspase-9 (38 kDa) (1:1000, CST, Boston, MA, USA), rabbit polyclonal against Bcl-2, mouse monoclonal against Bax antibodies (1:100, Santa Cruz Biotechnology), rabbit polyclonal against IL-1β receptor and IL-1β phosphorylation receptor (1:1000, CST), and rabbit polyclonal against TNF-α receptor I and TNF-α receptor I phosphorylation (1:1000, CST) for 2 h at room temperature and then with biotinylated goat anti-rabbit IgG (1: 200, Vector Labs) for 1 h at room temperature. .. The proteins were visualized using the ABC reagent (Vectastain ABC Kit, Vector Labs) and the ECL detection kit (Amersham Pharmacia Biotech, Baie-D’Urfe, QC, Canada).

    Article Title: Walnut Protein Hydrolysates Play a Protective Role on Neurotoxicity Induced by d-Galactose and Aluminum Chloride in Mice
    Article Snippet: .. After being blocked with a normal goat serum blocking solution for 20 min, the slices were incubated with primary antibodies of rabbit anti-TNF-α and anti-IL-1β (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. .. After washing three times with PBS, the slices were incubated with goat anti-rabbit secondary antibody for 20 min at 37 °C, and subsequently reacted with horseradish peroxidase-streptavidin (Streptavidin Peroxidase Link Detection Kits; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) for 20 min at room temperature.

    Western Blot:

    Article Title: Nickel induces inflammatory activation via NF-κB, MAPKs, IRF3 and NLRP3 inflammasome signaling pathways in macrophages
    Article Snippet: MitoSOX (M36008) was obtained from Invitrogen. .. The antibodies such as mouse anti-IκBα (#4814, 1:1000 for WB), rabbit anti-phospho (p)-IκBα (#2859, 1:1000 for WB), rabbit anti-NF-κB p65 (#8242, 1:1000 for WB, 1:200 for IF), mouse anti-β-Actin (#3700, 1:1000 for WB), rabbit anti-p38 (#8690, 1:1000 for WB), rabbit anti-p-p38 (#4511, 1:1000 for WB), rabbit anti-JNK (#9252, 1:1000 for WB), rabbit anti-p-JNK (#4668, 1:1000 for WB), rabbit anti-ERK (#4695, 1:1000 for WB), rabbit anti-p-ERK (#9101, 1:1000 for WB), rabbit anti-IRF3 (#11904, 1:1000 for WB), rabbit anti-p-IRF3 (#83611, 1:1000 for WB), rabbit anti-NLRP3 (#15101, 1:1000 for WB), rabbit anti-IL-1β (#12426S, 1:1000 for WB), rabbit anti-cleaved-caspase-3 (#9664, 1:1000 for WB), rabbit anti-cleaved-caspase-8 (#8592, 1:1000 for WB), rabbit anti-cleaved-caspase-9 (#9509, 1:1000 for WB), rabbit anti-PARP (#9548, 1:1000 for WB) were supplied by Cell Signaling Technology. .. Mouse anti-Histone H3 (sc-517576, 1:1000 for WB) and mouse anti-Tubulin (sc-73242, 1:1000 for WB) were obtained from Santa Cruz Biotechnology, while mouse anti-caspase-1 p20 (#AG-20B-0042-C100, 1:1000 for WB) and mouse anti-ASC (#AG-25B-0006-C100, 1:1000 for WB) were purchased from Adipogen.

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    Cell Signaling Technology Inc total p70s6k
    ST6Gal-I activity promotes the activation of prosurvival molecules upon acute serum deprivation. A , OV4 cells were serum-deprived for 4, 18, or 24 h and then immunoblotted for pAkt, total Akt, <t>p-p70S6K,</t> and total p70S6K. B , densitometric analyses for pAkt and total Akt for three blots. Values for pAkt and total Akt were each normalized to β-tubulin, and then data were plotted as pAkt/total Akt. *, p
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    Cell Signaling Technology Inc il 1β d3h1z rabbit mab mouse specific
    Increased IL1β secretion after the downregulation of Ubr-ubiquitin ligases of the Arg/N-degron pathway. ( a ) mRNA and ( b ) protein levels of UBR1, UBR2, UBR4, and UBR5 in J774A.1 cells after 72 h of exposure to 5 or 10-nM siRNA. ( c ) Western blot analysis of cleaved CASPASE-1 secreted in the media of J774A.1 cells treated with 10 nM of si-Ctrl or siRNA against Ubr (UBR1, UBR2, UBR4, and UBR5) for 72 h, followed by 1, 10, or 50 ng/mL of LPS for 6h and 5-mM ATP for the last hour. ( d ) Western blot analysis of pro- and cleaved <t>IL-1β</t> from the media and lysates of cells treated as in ( c ). ( e ) Quantification of the secreted pro- and cleaved IL-1β from the media of J774A.1 cells treated as in ( c ) by the cytokine bead assay (CBA) P -values were determined by a one-way ANOVA (** p
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    Cell Signaling Technology Inc il 1β
    Schematic indicating the suppression of microglial NLRP3 to alleviate morphine tolerance by procyanidins. Upon the stimulation by morphine or LPS, several pathways including the release of ROS, and K + efflux have been identified to signal the activation of NLRP3 inflammasome, leading to the activation of caspase-1 and the maturation of <t>IL-1β.</t> Procyanidins inhibited the ROS production and NLRP3 activation. The p38/NK-κB pathway was activated by morphine or LPS to induce the gene expression of NLRP3. The inhibition of p-p38 and NF-κB were a critical mechanism for the NLRP3 inflammasome suppressive effect of procyanidins in microglia
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    Cell Signaling Technology Inc rabbit polyclonal anti dyrk1a
    FINDY suppresses autophosphorylation of Ser97 in <t>DYRK1A.</t> ( a ) The bicistronic expression of intact DYRK1A (WT) and the substituted mutants of Lys188 to Arg (K188R), Ser97 to Ala (S97A), Tyr111 to Phe (Y111F), Tyr319 to Phe (Y319F), Tyr321 to Phe (Y321F), Tyr319 and Tyr321 to Phe (Y319F/Y321F) and Ser529 to Ala (S529A) with EGFP in HEK293 cells. The expression vectors were stably integrated in the genome of the cells. Each protein was detected with the corresponding antibody against FLAG, EGFP or GAPDH. Representative data from the quadruplicate experiments are shown. ( b ) The alignment of Ser97 and its surrounding amino-acid sequences from DYRK1A of the indicated species. The conserved serine residues corresponding to Ser97 of Homo sapiens DYRK1A are highlighted by the red rectangular box. Underlined portions indicate the phospho-peptide used in the immunization. ( c ) The <t>polyclonal</t> antibody against phosphorylated Ser97 (p-Ser97) specifically detected FLAG-DYRK1A (WT), but not the kinase-dead (K188R) or S97A mutants. The cells were transiently transfected with the expression vectors of the indicated constructs and the proteins were overexpressed. Total cell lysates were subjected to SDS–PAGE followed by western blot analysis. Representative data from the triplicate experiments are shown. ( d ) FINDY suppressed Ser97 autophosphorylation. FLAG-DYRK1A was produced in HEK293 cells incubated with epoxomicin and FINDY for 5 h, then purified with anti-FLAG antibody-conjugated beads. Each protein was detected with the corresponding antibodies against p-Ser97, phospho-Tyr (p-Tyr) or FLAG. Representative data from the triplicate experiments are shown. ( e ) FINDY did not suppress Ser97 autophosphorylation in Tc 2. FLAG-DYRK1A was produced in HEK293 cells for 16 h in the presence of doxycycline, after which cells were treated with 10 μM of FINDY or RD0392 for 5 h. Total cell lysates were subjected to immunoprecipitation (IP) with anti-FLAG antibody-conjugated beads. Each protein was detected with the corresponding antibodies against p-Ser97 and FLAG. Representative data from the duplicate experiments are shown. Conc., concentration.
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    ST6Gal-I activity promotes the activation of prosurvival molecules upon acute serum deprivation. A , OV4 cells were serum-deprived for 4, 18, or 24 h and then immunoblotted for pAkt, total Akt, p-p70S6K, and total p70S6K. B , densitometric analyses for pAkt and total Akt for three blots. Values for pAkt and total Akt were each normalized to β-tubulin, and then data were plotted as pAkt/total Akt. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: The Glycosyltransferase ST6Gal-I Protects Tumor Cells against Serum Growth Factor Withdrawal by Enhancing Survival Signaling and Proliferative Potential *

    doi: 10.1074/jbc.M116.763862

    Figure Lengend Snippet: ST6Gal-I activity promotes the activation of prosurvival molecules upon acute serum deprivation. A , OV4 cells were serum-deprived for 4, 18, or 24 h and then immunoblotted for pAkt, total Akt, p-p70S6K, and total p70S6K. B , densitometric analyses for pAkt and total Akt for three blots. Values for pAkt and total Akt were each normalized to β-tubulin, and then data were plotted as pAkt/total Akt. *, p

    Article Snippet: Immunoblots were probed with antibodies to ST6Gal-I (R & D Systems, AF5924), cIAP2 (Cell Signaling Technology, 3130, lot 6), pAkt (Ser-473, Cell Signaling Technology, 4060, lot 19), total Akt (Cell Signaling Technology, 4691, lot 20), AIF (Cell Signaling Technology, 4642, lot 3), Beclin (Cell Signaling Technology, 3495, lot 2), p-p70S6K (Thr-389, Cell Signaling Technology, 9234, lot 11), total p70S6K (Cell Signaling Technology, 2708, lot 7), pNFκB (p65, Ser-536, Cell Signaling Technology, 3033, lot 14), total NFκB (p65, Cell Signaling Technology, 8242, lot 4), cyclin D2 (Cell Signaling Technology, 3741, lot 4), p-pRb (Ser-809/811, Cell Signaling Technology, 8516, lot 4), or total pRb (Cell Signaling Technology, 9309, lot 9).

    Techniques: Activity Assay, Activation Assay

    ST6Gal-I knockdown inhibits activation of prosurvival molecules upon acute serum deprivation. A , BxPC3 cells were serum-deprived for 4, 18, or 24 h and then immunoblotted for pAkt, total Akt, p-p70S6K, and total p70S6K. B , densitometric analyses for pAkt and total Akt from three blots. Values for pAkt and total Akt were each normalized to β-tubulin, and then data were plotted as pAkt/total Akt. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: The Glycosyltransferase ST6Gal-I Protects Tumor Cells against Serum Growth Factor Withdrawal by Enhancing Survival Signaling and Proliferative Potential *

    doi: 10.1074/jbc.M116.763862

    Figure Lengend Snippet: ST6Gal-I knockdown inhibits activation of prosurvival molecules upon acute serum deprivation. A , BxPC3 cells were serum-deprived for 4, 18, or 24 h and then immunoblotted for pAkt, total Akt, p-p70S6K, and total p70S6K. B , densitometric analyses for pAkt and total Akt from three blots. Values for pAkt and total Akt were each normalized to β-tubulin, and then data were plotted as pAkt/total Akt. *, p

    Article Snippet: Immunoblots were probed with antibodies to ST6Gal-I (R & D Systems, AF5924), cIAP2 (Cell Signaling Technology, 3130, lot 6), pAkt (Ser-473, Cell Signaling Technology, 4060, lot 19), total Akt (Cell Signaling Technology, 4691, lot 20), AIF (Cell Signaling Technology, 4642, lot 3), Beclin (Cell Signaling Technology, 3495, lot 2), p-p70S6K (Thr-389, Cell Signaling Technology, 9234, lot 11), total p70S6K (Cell Signaling Technology, 2708, lot 7), pNFκB (p65, Ser-536, Cell Signaling Technology, 3033, lot 14), total NFκB (p65, Cell Signaling Technology, 8242, lot 4), cyclin D2 (Cell Signaling Technology, 3741, lot 4), p-pRb (Ser-809/811, Cell Signaling Technology, 8516, lot 4), or total pRb (Cell Signaling Technology, 9309, lot 9).

    Techniques: Activation Assay

    Increased IL1β secretion after the downregulation of Ubr-ubiquitin ligases of the Arg/N-degron pathway. ( a ) mRNA and ( b ) protein levels of UBR1, UBR2, UBR4, and UBR5 in J774A.1 cells after 72 h of exposure to 5 or 10-nM siRNA. ( c ) Western blot analysis of cleaved CASPASE-1 secreted in the media of J774A.1 cells treated with 10 nM of si-Ctrl or siRNA against Ubr (UBR1, UBR2, UBR4, and UBR5) for 72 h, followed by 1, 10, or 50 ng/mL of LPS for 6h and 5-mM ATP for the last hour. ( d ) Western blot analysis of pro- and cleaved IL-1β from the media and lysates of cells treated as in ( c ). ( e ) Quantification of the secreted pro- and cleaved IL-1β from the media of J774A.1 cells treated as in ( c ) by the cytokine bead assay (CBA) P -values were determined by a one-way ANOVA (** p

    Journal: Biomolecules

    Article Title: The Arg/N-Degron Pathway—A Potential Running Back in Fine-Tuning the Inflammatory Response?

    doi: 10.3390/biom10060903

    Figure Lengend Snippet: Increased IL1β secretion after the downregulation of Ubr-ubiquitin ligases of the Arg/N-degron pathway. ( a ) mRNA and ( b ) protein levels of UBR1, UBR2, UBR4, and UBR5 in J774A.1 cells after 72 h of exposure to 5 or 10-nM siRNA. ( c ) Western blot analysis of cleaved CASPASE-1 secreted in the media of J774A.1 cells treated with 10 nM of si-Ctrl or siRNA against Ubr (UBR1, UBR2, UBR4, and UBR5) for 72 h, followed by 1, 10, or 50 ng/mL of LPS for 6h and 5-mM ATP for the last hour. ( d ) Western blot analysis of pro- and cleaved IL-1β from the media and lysates of cells treated as in ( c ). ( e ) Quantification of the secreted pro- and cleaved IL-1β from the media of J774A.1 cells treated as in ( c ) by the cytokine bead assay (CBA) P -values were determined by a one-way ANOVA (** p

    Article Snippet: PAGE-fractionated proteins were transferred onto the nitrocellulose membrane and analyzed by Western blot using the following antibodies: anti-UBR1 (Abcam, Cambridge, MA, USA, #156436), anti-UBR2 (Abcam #191505), anti-UBR4 (Abcam #86738), anti-EDD (Santa Cruz, Dallas, TX, USA, #515494), anti-GAPDH (Santa Cruz #sc32233), anti- IL-1β (Cell Signaling, Danvers, MA, USA, #12507), anticleaved-caspase-1 (P20) (Cell Signaling #4199), and anti-caspase-3 (Cell Signaling #14220).

    Techniques: Western Blot, Crocin Bleaching Assay

    Schematic indicating the suppression of microglial NLRP3 to alleviate morphine tolerance by procyanidins. Upon the stimulation by morphine or LPS, several pathways including the release of ROS, and K + efflux have been identified to signal the activation of NLRP3 inflammasome, leading to the activation of caspase-1 and the maturation of IL-1β. Procyanidins inhibited the ROS production and NLRP3 activation. The p38/NK-κB pathway was activated by morphine or LPS to induce the gene expression of NLRP3. The inhibition of p-p38 and NF-κB were a critical mechanism for the NLRP3 inflammasome suppressive effect of procyanidins in microglia

    Journal: Journal of Neuroinflammation

    Article Title: Procyanidins alleviates morphine tolerance by inhibiting activation of NLRP3 inflammasome in microglia

    doi: 10.1186/s12974-016-0520-z

    Figure Lengend Snippet: Schematic indicating the suppression of microglial NLRP3 to alleviate morphine tolerance by procyanidins. Upon the stimulation by morphine or LPS, several pathways including the release of ROS, and K + efflux have been identified to signal the activation of NLRP3 inflammasome, leading to the activation of caspase-1 and the maturation of IL-1β. Procyanidins inhibited the ROS production and NLRP3 activation. The p38/NK-κB pathway was activated by morphine or LPS to induce the gene expression of NLRP3. The inhibition of p-p38 and NF-κB were a critical mechanism for the NLRP3 inflammasome suppressive effect of procyanidins in microglia

    Article Snippet: The membranes were blocked with 10 % whole milk in TBST (Tris–HCl, NaCl, Tween 20) for 2 h at room temperature, probed with primary antibodies at 4 °C overnight [GAPDH, 1:8000; IBA-1, 1:1000; NLRP3, 1;1000; caspase-1, 1:1000; IL-1β, 1:500; TNF-α, 1:1000; p-p38 (Tyr182), 1:1000; p38, 1:1000; p-ERK, 1:1000; ERK, 1:1000; p-JNK, 1:1000; JNK, 1:1000; p-NR1(Ser896), 1:1000; NR1, 1:1000; p-PKC, 1:1000; PKC, 1:1000;] and then incubated with horseradish peroxidase-coupled secondary antibodies from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Activation Assay, Expressing, Inhibition

    Procyanidins suppressed morphine-induced NLRP3 inflammasome activation in the spinal cord. a Procyanidins inhibited the morphine-induced upregulation of IL-1β in the spinal cord. b Procyanidins suppressed the morphine-induced activation of caspase-1 in the spinal cord. c Procyanidins inhibited the morphine-induced upregulation of NLRP3 in the spinal cord. d Procyanidins inhibited the morphine-induced upregulation of TNF-α in the spinal cord. The western blot samples ( n = 4) were collected as described in methods. e Procyanidins inhibited the morphine-induced upregulation of malondialdehyde (MDA) in the spinal cord ( n = 4). * p

    Journal: Journal of Neuroinflammation

    Article Title: Procyanidins alleviates morphine tolerance by inhibiting activation of NLRP3 inflammasome in microglia

    doi: 10.1186/s12974-016-0520-z

    Figure Lengend Snippet: Procyanidins suppressed morphine-induced NLRP3 inflammasome activation in the spinal cord. a Procyanidins inhibited the morphine-induced upregulation of IL-1β in the spinal cord. b Procyanidins suppressed the morphine-induced activation of caspase-1 in the spinal cord. c Procyanidins inhibited the morphine-induced upregulation of NLRP3 in the spinal cord. d Procyanidins inhibited the morphine-induced upregulation of TNF-α in the spinal cord. The western blot samples ( n = 4) were collected as described in methods. e Procyanidins inhibited the morphine-induced upregulation of malondialdehyde (MDA) in the spinal cord ( n = 4). * p

    Article Snippet: The membranes were blocked with 10 % whole milk in TBST (Tris–HCl, NaCl, Tween 20) for 2 h at room temperature, probed with primary antibodies at 4 °C overnight [GAPDH, 1:8000; IBA-1, 1:1000; NLRP3, 1;1000; caspase-1, 1:1000; IL-1β, 1:500; TNF-α, 1:1000; p-p38 (Tyr182), 1:1000; p38, 1:1000; p-ERK, 1:1000; ERK, 1:1000; p-JNK, 1:1000; JNK, 1:1000; p-NR1(Ser896), 1:1000; NR1, 1:1000; p-PKC, 1:1000; PKC, 1:1000;] and then incubated with horseradish peroxidase-coupled secondary antibodies from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Activation Assay, Western Blot, Multiple Displacement Amplification

    Procyanidins suppressed morphine-induced NLRP3 inflammasome activation in microglia. BV-2 cells were stimulated by LPS (1 μg/ml) or morphine (200 μM) with or without 100 μM of procyanidins for 12 h, and then, the inflammasome was activated with 5 mM of ATP for 1 h. a Procyanidins (100 μM) inhibited both LPS-induced and morphine-induced expression of pro-IL-1β and NLRP3 in BV-2 cells. The western blot samples for BV-2 cells ( n = 4) were collected and analyzed 12 h after the last morphine exposure. b Procyanidins (100 μM) inhibited the morphine-induced and LPS-induced increase of caspase-1 and IL-1β in the supernatant of BV-2 cells. The western blot samples ( n = 4) came from the supernatant of BV-2 cells. c ELISA of IL-1β in supernatants from LPS- and morphine-primed BV-2 cells and then stimulated with ATP. * p

    Journal: Journal of Neuroinflammation

    Article Title: Procyanidins alleviates morphine tolerance by inhibiting activation of NLRP3 inflammasome in microglia

    doi: 10.1186/s12974-016-0520-z

    Figure Lengend Snippet: Procyanidins suppressed morphine-induced NLRP3 inflammasome activation in microglia. BV-2 cells were stimulated by LPS (1 μg/ml) or morphine (200 μM) with or without 100 μM of procyanidins for 12 h, and then, the inflammasome was activated with 5 mM of ATP for 1 h. a Procyanidins (100 μM) inhibited both LPS-induced and morphine-induced expression of pro-IL-1β and NLRP3 in BV-2 cells. The western blot samples for BV-2 cells ( n = 4) were collected and analyzed 12 h after the last morphine exposure. b Procyanidins (100 μM) inhibited the morphine-induced and LPS-induced increase of caspase-1 and IL-1β in the supernatant of BV-2 cells. The western blot samples ( n = 4) came from the supernatant of BV-2 cells. c ELISA of IL-1β in supernatants from LPS- and morphine-primed BV-2 cells and then stimulated with ATP. * p

    Article Snippet: The membranes were blocked with 10 % whole milk in TBST (Tris–HCl, NaCl, Tween 20) for 2 h at room temperature, probed with primary antibodies at 4 °C overnight [GAPDH, 1:8000; IBA-1, 1:1000; NLRP3, 1;1000; caspase-1, 1:1000; IL-1β, 1:500; TNF-α, 1:1000; p-p38 (Tyr182), 1:1000; p38, 1:1000; p-ERK, 1:1000; ERK, 1:1000; p-JNK, 1:1000; JNK, 1:1000; p-NR1(Ser896), 1:1000; NR1, 1:1000; p-PKC, 1:1000; PKC, 1:1000;] and then incubated with horseradish peroxidase-coupled secondary antibodies from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Activation Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

    FINDY suppresses autophosphorylation of Ser97 in DYRK1A. ( a ) The bicistronic expression of intact DYRK1A (WT) and the substituted mutants of Lys188 to Arg (K188R), Ser97 to Ala (S97A), Tyr111 to Phe (Y111F), Tyr319 to Phe (Y319F), Tyr321 to Phe (Y321F), Tyr319 and Tyr321 to Phe (Y319F/Y321F) and Ser529 to Ala (S529A) with EGFP in HEK293 cells. The expression vectors were stably integrated in the genome of the cells. Each protein was detected with the corresponding antibody against FLAG, EGFP or GAPDH. Representative data from the quadruplicate experiments are shown. ( b ) The alignment of Ser97 and its surrounding amino-acid sequences from DYRK1A of the indicated species. The conserved serine residues corresponding to Ser97 of Homo sapiens DYRK1A are highlighted by the red rectangular box. Underlined portions indicate the phospho-peptide used in the immunization. ( c ) The polyclonal antibody against phosphorylated Ser97 (p-Ser97) specifically detected FLAG-DYRK1A (WT), but not the kinase-dead (K188R) or S97A mutants. The cells were transiently transfected with the expression vectors of the indicated constructs and the proteins were overexpressed. Total cell lysates were subjected to SDS–PAGE followed by western blot analysis. Representative data from the triplicate experiments are shown. ( d ) FINDY suppressed Ser97 autophosphorylation. FLAG-DYRK1A was produced in HEK293 cells incubated with epoxomicin and FINDY for 5 h, then purified with anti-FLAG antibody-conjugated beads. Each protein was detected with the corresponding antibodies against p-Ser97, phospho-Tyr (p-Tyr) or FLAG. Representative data from the triplicate experiments are shown. ( e ) FINDY did not suppress Ser97 autophosphorylation in Tc 2. FLAG-DYRK1A was produced in HEK293 cells for 16 h in the presence of doxycycline, after which cells were treated with 10 μM of FINDY or RD0392 for 5 h. Total cell lysates were subjected to immunoprecipitation (IP) with anti-FLAG antibody-conjugated beads. Each protein was detected with the corresponding antibodies against p-Ser97 and FLAG. Representative data from the duplicate experiments are shown. Conc., concentration.

    Journal: Nature Communications

    Article Title: Selective inhibition of the kinase DYRK1A by targeting its folding process

    doi: 10.1038/ncomms11391

    Figure Lengend Snippet: FINDY suppresses autophosphorylation of Ser97 in DYRK1A. ( a ) The bicistronic expression of intact DYRK1A (WT) and the substituted mutants of Lys188 to Arg (K188R), Ser97 to Ala (S97A), Tyr111 to Phe (Y111F), Tyr319 to Phe (Y319F), Tyr321 to Phe (Y321F), Tyr319 and Tyr321 to Phe (Y319F/Y321F) and Ser529 to Ala (S529A) with EGFP in HEK293 cells. The expression vectors were stably integrated in the genome of the cells. Each protein was detected with the corresponding antibody against FLAG, EGFP or GAPDH. Representative data from the quadruplicate experiments are shown. ( b ) The alignment of Ser97 and its surrounding amino-acid sequences from DYRK1A of the indicated species. The conserved serine residues corresponding to Ser97 of Homo sapiens DYRK1A are highlighted by the red rectangular box. Underlined portions indicate the phospho-peptide used in the immunization. ( c ) The polyclonal antibody against phosphorylated Ser97 (p-Ser97) specifically detected FLAG-DYRK1A (WT), but not the kinase-dead (K188R) or S97A mutants. The cells were transiently transfected with the expression vectors of the indicated constructs and the proteins were overexpressed. Total cell lysates were subjected to SDS–PAGE followed by western blot analysis. Representative data from the triplicate experiments are shown. ( d ) FINDY suppressed Ser97 autophosphorylation. FLAG-DYRK1A was produced in HEK293 cells incubated with epoxomicin and FINDY for 5 h, then purified with anti-FLAG antibody-conjugated beads. Each protein was detected with the corresponding antibodies against p-Ser97, phospho-Tyr (p-Tyr) or FLAG. Representative data from the triplicate experiments are shown. ( e ) FINDY did not suppress Ser97 autophosphorylation in Tc 2. FLAG-DYRK1A was produced in HEK293 cells for 16 h in the presence of doxycycline, after which cells were treated with 10 μM of FINDY or RD0392 for 5 h. Total cell lysates were subjected to immunoprecipitation (IP) with anti-FLAG antibody-conjugated beads. Each protein was detected with the corresponding antibodies against p-Ser97 and FLAG. Representative data from the duplicate experiments are shown. Conc., concentration.

    Article Snippet: Rabbit polyclonal anti-DYRK1A (1/3,000, #2771S/Lot: 1), p70S6K (1/3,000, #9202S/Lot: 8), CHK2 (1/3,000, #2662S/Lot: 5), MARK3 (1/3,000, #9311BC/Lot: 1), RAF1 (1/3,000, #9422BC/Lot: 5), FAK (1/3,000, #3285BC/Lot: 6), FYN (1/3,000, #4023BC/Lot: 2) and ABL (1/3,000, #2862BC/Lot: 11) antibodies were obtained from Cell Signaling Technology.

    Techniques: Expressing, Stable Transfection, Transfection, Construct, SDS Page, Western Blot, Produced, Incubation, Purification, Immunoprecipitation, Concentration Assay