recombinant human histones h3  (New England Biolabs)


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    Name:
    Histone H3 3 Human Recombinant
    Description:
    Histone H3 3 Human Recombinant 100 ug
    Catalog Number:
    m2507s
    Price:
    82
    Size:
    100 ug
    Category:
    DNA Binding Proteins
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    New England Biolabs recombinant human histones h3
    Histone H3 3 Human Recombinant
    Histone H3 3 Human Recombinant 100 ug
    https://www.bioz.com/result/recombinant human histones h3/product/New England Biolabs
    Average 94 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    recombinant human histones h3 - by Bioz Stars, 2020-09
    94/100 stars

    Images

    1) Product Images from "In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps"

    Article Title: In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps

    Journal: Blood

    doi: 10.1182/blood-2016-06-722298

    Individual human histones H3 and H4, but not octameric core histones, trigger TG in PRP. TG performed in recalcified PRP containing individual human histone proteins was compared with that of recombinant core histone octamers (A) or purified calf thymus histones (B). CThist, mixture of purified calf thymus histones; rOctamer, octameric core histone reconstituted with recombinant human histone proteins. Panels A and B are representative of 3 independent experiments.
    Figure Legend Snippet: Individual human histones H3 and H4, but not octameric core histones, trigger TG in PRP. TG performed in recalcified PRP containing individual human histone proteins was compared with that of recombinant core histone octamers (A) or purified calf thymus histones (B). CThist, mixture of purified calf thymus histones; rOctamer, octameric core histone reconstituted with recombinant human histone proteins. Panels A and B are representative of 3 independent experiments.

    Techniques Used: Recombinant, Purification

    hnDNA and individual human histone H3 and H4 trigger coagulation in plasma. TG in recalcified normal PFP (A) and normal PRP (B) containing hnDNA. TG in recalcified FXII-deficient (FXII-Def), FXI-deficient (FXI-Def), or FVII-deficient (FVII-Def) PFP in the presence or absence of 30 µg/mL of hnDNA (C). No TG was observed in any individual deficient plasma after recalcification in the absence of DNA, represented by a single flat curve (PFP no DNA, panel C). Quantification of FXIa-AT after activation of the contact system by hnDNA in the synthetic contact system activation assay as described in “Methods” (D). TG in recalcified PRP containing recombinant human histone H3 (rH3) (E) or recombinant human histone H4 (rH4) (F). Effect of citrullination of histones H3 (G) and H4 (H) on TG in recalcified PRP. All the figures are representative of at least 3 independent experiments.
    Figure Legend Snippet: hnDNA and individual human histone H3 and H4 trigger coagulation in plasma. TG in recalcified normal PFP (A) and normal PRP (B) containing hnDNA. TG in recalcified FXII-deficient (FXII-Def), FXI-deficient (FXI-Def), or FVII-deficient (FVII-Def) PFP in the presence or absence of 30 µg/mL of hnDNA (C). No TG was observed in any individual deficient plasma after recalcification in the absence of DNA, represented by a single flat curve (PFP no DNA, panel C). Quantification of FXIa-AT after activation of the contact system by hnDNA in the synthetic contact system activation assay as described in “Methods” (D). TG in recalcified PRP containing recombinant human histone H3 (rH3) (E) or recombinant human histone H4 (rH4) (F). Effect of citrullination of histones H3 (G) and H4 (H) on TG in recalcified PRP. All the figures are representative of at least 3 independent experiments.

    Techniques Used: Coagulation, Activation Assay, Recombinant

    2) Product Images from "A functional SUMO-motif in the active site of PIM1 promotes its degradation via RNF4, and stimulates protein kinase activity"

    Article Title: A functional SUMO-motif in the active site of PIM1 promotes its degradation via RNF4, and stimulates protein kinase activity

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-03775-w

    SUMOylation increase PIM1 kinase activity in vitro . ( a ) Bacterially purified 6His-PIM1 was SUMOylated in vitro using purified GST-SUMO2. Equal amounts of SUMOylated protein (including PIM1) were captured using GST-beads and incubated without or with SENP1 catalytic domain for 1 hour at 30 °C. Kinase assays were then performed using Histone H3.3 as a substrate for at 30 °C for 0, 15, 30 and 45 min. Kinase activity of SUMO2-modified or unmodified PIM1 was measured by analyzing Histone H3.3 phosphorylation using a phospho-specific antibody. Equal levels of substrate and kinase were confirmed by western blotting using indicated antibodies. ( b ) Purified WT PIM1 was first incubated with or without SENP1 catalytic domain fragment for 1 hour at 30 °C, and immediately used in a kinase assay using Histone H3.3 as substrate for 30 min at 30 °C. PIM1 kinase activity was measured by analyzing Histone H3.3 phosphorylation using a phospho-specific antibody. Equal levels of substrate and kinase were confirmed by coomassie staining of the gel.
    Figure Legend Snippet: SUMOylation increase PIM1 kinase activity in vitro . ( a ) Bacterially purified 6His-PIM1 was SUMOylated in vitro using purified GST-SUMO2. Equal amounts of SUMOylated protein (including PIM1) were captured using GST-beads and incubated without or with SENP1 catalytic domain for 1 hour at 30 °C. Kinase assays were then performed using Histone H3.3 as a substrate for at 30 °C for 0, 15, 30 and 45 min. Kinase activity of SUMO2-modified or unmodified PIM1 was measured by analyzing Histone H3.3 phosphorylation using a phospho-specific antibody. Equal levels of substrate and kinase were confirmed by western blotting using indicated antibodies. ( b ) Purified WT PIM1 was first incubated with or without SENP1 catalytic domain fragment for 1 hour at 30 °C, and immediately used in a kinase assay using Histone H3.3 as substrate for 30 min at 30 °C. PIM1 kinase activity was measured by analyzing Histone H3.3 phosphorylation using a phospho-specific antibody. Equal levels of substrate and kinase were confirmed by coomassie staining of the gel.

    Techniques Used: Activity Assay, In Vitro, Purification, Incubation, Modification, Western Blot, Kinase Assay, Staining

    PIM1 SUMOylation regulates substrate specificity in vitro and in cultured cells. ( a ) 6His-PIM1 (WT or mutant) was expressed and purified from bacterial cells, and resolved by SDS-PAGE. A western blot for the same samples was also performed using a pan-phospho tyrosine antibody to detect PIM1 autophosphorylation. ( b ) The purified 6His-PIM1 proteins were treated with lambda phosphatase (+) to remove overall phosphorylation or untreated (−). Samples were resolved by SDS-PAGE, and stained with coomassie to visualize a shift in mobility, which is indicative of dephosphorylation. ( c ) In vitro kinase assays were carried out using recombinant c-MYC or Histone H3.3 as substrates, in the absence or presence of the indicated purified 6His-PIM1 proteins. The samples were resolved by SDS-PAGE, and either stained with coomassie to detect total protein levels or transferred to a nitrocellulose membrane for western blotting using phospho-specific antibodies as a measure of PIM1 kinase activity. ( d ) U2OS-FRT cells expressing YFP alone, YFP-WT PIM1 and YFP-E171A were treated with 10 ng/ml doxycycline; U2OS-FRT expressing YFP-K169R was treated with 20 ng/ml doxycycline and U2OS-FRT expressing YFP-K67M was treated with 50 ng/ml doxycycline for 48 hours, followed by western blotting using indicated antibodies.
    Figure Legend Snippet: PIM1 SUMOylation regulates substrate specificity in vitro and in cultured cells. ( a ) 6His-PIM1 (WT or mutant) was expressed and purified from bacterial cells, and resolved by SDS-PAGE. A western blot for the same samples was also performed using a pan-phospho tyrosine antibody to detect PIM1 autophosphorylation. ( b ) The purified 6His-PIM1 proteins were treated with lambda phosphatase (+) to remove overall phosphorylation or untreated (−). Samples were resolved by SDS-PAGE, and stained with coomassie to visualize a shift in mobility, which is indicative of dephosphorylation. ( c ) In vitro kinase assays were carried out using recombinant c-MYC or Histone H3.3 as substrates, in the absence or presence of the indicated purified 6His-PIM1 proteins. The samples were resolved by SDS-PAGE, and either stained with coomassie to detect total protein levels or transferred to a nitrocellulose membrane for western blotting using phospho-specific antibodies as a measure of PIM1 kinase activity. ( d ) U2OS-FRT cells expressing YFP alone, YFP-WT PIM1 and YFP-E171A were treated with 10 ng/ml doxycycline; U2OS-FRT expressing YFP-K169R was treated with 20 ng/ml doxycycline and U2OS-FRT expressing YFP-K67M was treated with 50 ng/ml doxycycline for 48 hours, followed by western blotting using indicated antibodies.

    Techniques Used: In Vitro, Cell Culture, Mutagenesis, Purification, SDS Page, Western Blot, Staining, De-Phosphorylation Assay, Recombinant, Activity Assay, Expressing

    3) Product Images from "RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome"

    Article Title: RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome

    Journal: Nature Communications

    doi: 10.1038/s41467-018-08030-y

    Efficacy of anti-histone aptamers when administered after histones. a Aptamer inhibition of histone-mediated cytotoxicity of endothelial cells determined by MTS assay. EA.hy926 cells treated with 200 µg mL −1 of calf thymus histones followed by administration of either vehicle (negative control), heparin (positive control, 1:1), KU7 aptamer (1:2) or KU9 aptamer (1:2) at time points of 0, 5, 10, 15, 30, 45, 60, 90, 120 and 180 min after CTH; n = 3 biological replicates; * p
    Figure Legend Snippet: Efficacy of anti-histone aptamers when administered after histones. a Aptamer inhibition of histone-mediated cytotoxicity of endothelial cells determined by MTS assay. EA.hy926 cells treated with 200 µg mL −1 of calf thymus histones followed by administration of either vehicle (negative control), heparin (positive control, 1:1), KU7 aptamer (1:2) or KU9 aptamer (1:2) at time points of 0, 5, 10, 15, 30, 45, 60, 90, 120 and 180 min after CTH; n = 3 biological replicates; * p

    Techniques Used: Inhibition, MTS Assay, Negative Control, Positive Control

    Aptamers bind to human neutrophil-derived NETs and inhibit NET-induced cytotoxicity. a Confocal microscopy of human neutrophil-derived NETs. Single images are shown in gray scale. DAPI (top, left); histones (top, right); aptamer KU7 (bottom, left); merged images show: cyan, DAPI labeling of DNA, yellow, histones, and magenta, aptamer KU7–647 (bottom, right). White areas in the merged image represent close proximity of DNA, histone and aptamer. Representative images, captured with 40× oil and 2.8× zoom. Scale is equivalent to 10 µm. b Aptamer inhibition of NETs-mediated cytotoxicity of endothelial cells determined by MTS assay. EA.hy926 cells treated with 8 µg per well of NETs material (based on DNA concentration) and/or 8 µg per well (10.66 µM) of aptamer (KU7); * p
    Figure Legend Snippet: Aptamers bind to human neutrophil-derived NETs and inhibit NET-induced cytotoxicity. a Confocal microscopy of human neutrophil-derived NETs. Single images are shown in gray scale. DAPI (top, left); histones (top, right); aptamer KU7 (bottom, left); merged images show: cyan, DAPI labeling of DNA, yellow, histones, and magenta, aptamer KU7–647 (bottom, right). White areas in the merged image represent close proximity of DNA, histone and aptamer. Representative images, captured with 40× oil and 2.8× zoom. Scale is equivalent to 10 µm. b Aptamer inhibition of NETs-mediated cytotoxicity of endothelial cells determined by MTS assay. EA.hy926 cells treated with 8 µg per well of NETs material (based on DNA concentration) and/or 8 µg per well (10.66 µM) of aptamer (KU7); * p

    Techniques Used: Derivative Assay, Confocal Microscopy, Labeling, Inhibition, MTS Assay, Concentration Assay

    Identification of histone-specific RNA aptamers using SELEX. a Schematic of the in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX) procedure. Step 1. Double-stranded DNA (DS DNA) template library (Sel2N20) is in vitro transcribed in the presence of 2′ Fluoro pyrimidines and 2’ OH purines to generate the 2′ Fluoro-modified Round 0 RNA library (RNA). Step 2. The round 0 RNA library was incubated with human albumin and human IgG to remove RNAs that bind to human serum proteins (Negative selection). Step 3. RNA bound to serum proteins was discarded. Step 4. RNA not bound to serum proteins was incubated with human histones H3 and H4, respectively. Step 5. Histone-bound aptamers were collected and reverse-transcribed into DNA. Step 6. Round 1 DNA was then transcribed into RNA for the subsequent round of selection. A total of eight rounds of selection were performed for each histone selection (see Supplementary Table 1 ). b Binding of Round 0 (R0) and Round 8 (R8) RNA to recombinant human histone H3 (top, left panel) and H4 (top, right panel) proteins. Binding of R8 RNA to human albumin (bottom, left panel) and human serum (bottom, right panel). c Percent sequence enrichment (% Enrichment) at each round of selection (black circle). The 50% sequence enrichment point (gray circle) is indicated for each selection
    Figure Legend Snippet: Identification of histone-specific RNA aptamers using SELEX. a Schematic of the in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX) procedure. Step 1. Double-stranded DNA (DS DNA) template library (Sel2N20) is in vitro transcribed in the presence of 2′ Fluoro pyrimidines and 2’ OH purines to generate the 2′ Fluoro-modified Round 0 RNA library (RNA). Step 2. The round 0 RNA library was incubated with human albumin and human IgG to remove RNAs that bind to human serum proteins (Negative selection). Step 3. RNA bound to serum proteins was discarded. Step 4. RNA not bound to serum proteins was incubated with human histones H3 and H4, respectively. Step 5. Histone-bound aptamers were collected and reverse-transcribed into DNA. Step 6. Round 1 DNA was then transcribed into RNA for the subsequent round of selection. A total of eight rounds of selection were performed for each histone selection (see Supplementary Table 1 ). b Binding of Round 0 (R0) and Round 8 (R8) RNA to recombinant human histone H3 (top, left panel) and H4 (top, right panel) proteins. Binding of R8 RNA to human albumin (bottom, left panel) and human serum (bottom, right panel). c Percent sequence enrichment (% Enrichment) at each round of selection (black circle). The 50% sequence enrichment point (gray circle) is indicated for each selection

    Techniques Used: In Vitro, Modification, Incubation, Selection, Binding Assay, Recombinant, Sequencing

    Binding characterization and stability measurements of individual histone RNA aptamer sequences. a Binding kinetic rate constants ( k a and K D ) determined for aptamers KU7 (left panels) and KU9 (right panels) binding to CTH (top panels), H4 (middle panels) and BSA (bottom panels). Aptamers concentrations tested: 100 nM (blue), 50 nM (black), 25 nM (red), 12.5 nM (green), 10 nM (magenta). b Serum stability measurements for aptamers KU7 and KU9 (5 μM) in 50% human serum. T 1/2 KU7 = 150 h. T 1/2 KU9 = 48 h
    Figure Legend Snippet: Binding characterization and stability measurements of individual histone RNA aptamer sequences. a Binding kinetic rate constants ( k a and K D ) determined for aptamers KU7 (left panels) and KU9 (right panels) binding to CTH (top panels), H4 (middle panels) and BSA (bottom panels). Aptamers concentrations tested: 100 nM (blue), 50 nM (black), 25 nM (red), 12.5 nM (green), 10 nM (magenta). b Serum stability measurements for aptamers KU7 and KU9 (5 μM) in 50% human serum. T 1/2 KU7 = 150 h. T 1/2 KU9 = 48 h

    Techniques Used: Binding Assay

    Efficacy of histone aptamer in murine model of MODS. a Survival curves of mice injected IV with CTH in the presence or absence of aptamer treatment; n = 6 per group. Molar ratio of CTH to aptamer indicated. b Weights of the liver, lung and spleen normalized to pre-treatment body weight; * p
    Figure Legend Snippet: Efficacy of histone aptamer in murine model of MODS. a Survival curves of mice injected IV with CTH in the presence or absence of aptamer treatment; n = 6 per group. Molar ratio of CTH to aptamer indicated. b Weights of the liver, lung and spleen normalized to pre-treatment body weight; * p

    Techniques Used: Mouse Assay, Injection

    In vitro efficacy of RNA aptamers. a Human platelet aggregation measurements using platelets derived from three independent healthy donors. Collagen (Col), histone H4 (H4), histone aptamers (KU7 and KU9), calf thymus histones (CTH), heparin (Hep); * p
    Figure Legend Snippet: In vitro efficacy of RNA aptamers. a Human platelet aggregation measurements using platelets derived from three independent healthy donors. Collagen (Col), histone H4 (H4), histone aptamers (KU7 and KU9), calf thymus histones (CTH), heparin (Hep); * p

    Techniques Used: In Vitro, Derivative Assay

    4) Product Images from "RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome"

    Article Title: RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome

    Journal: Nature Communications

    doi: 10.1038/s41467-018-08030-y

    Efficacy of anti-histone aptamers when administered after histones. a Aptamer inhibition of histone-mediated cytotoxicity of endothelial cells determined by MTS assay. EA.hy926 cells treated with 200 µg mL −1 of calf thymus histones followed by administration of either vehicle (negative control), heparin (positive control, 1:1), KU7 aptamer (1:2) or KU9 aptamer (1:2) at time points of 0, 5, 10, 15, 30, 45, 60, 90, 120 and 180 min after CTH; n = 3 biological replicates; * p
    Figure Legend Snippet: Efficacy of anti-histone aptamers when administered after histones. a Aptamer inhibition of histone-mediated cytotoxicity of endothelial cells determined by MTS assay. EA.hy926 cells treated with 200 µg mL −1 of calf thymus histones followed by administration of either vehicle (negative control), heparin (positive control, 1:1), KU7 aptamer (1:2) or KU9 aptamer (1:2) at time points of 0, 5, 10, 15, 30, 45, 60, 90, 120 and 180 min after CTH; n = 3 biological replicates; * p

    Techniques Used: Inhibition, MTS Assay, Negative Control, Positive Control

    Aptamers bind to human neutrophil-derived NETs and inhibit NET-induced cytotoxicity. a Confocal microscopy of human neutrophil-derived NETs. Single images are shown in gray scale. DAPI (top, left); histones (top, right); aptamer KU7 (bottom, left); merged images show: cyan, DAPI labeling of DNA, yellow, histones, and magenta, aptamer KU7–647 (bottom, right). White areas in the merged image represent close proximity of DNA, histone and aptamer. Representative images, captured with 40× oil and 2.8× zoom. Scale is equivalent to 10 µm. b Aptamer inhibition of NETs-mediated cytotoxicity of endothelial cells determined by MTS assay. EA.hy926 cells treated with 8 µg per well of NETs material (based on DNA concentration) and/or 8 µg per well (10.66 µM) of aptamer (KU7); * p
    Figure Legend Snippet: Aptamers bind to human neutrophil-derived NETs and inhibit NET-induced cytotoxicity. a Confocal microscopy of human neutrophil-derived NETs. Single images are shown in gray scale. DAPI (top, left); histones (top, right); aptamer KU7 (bottom, left); merged images show: cyan, DAPI labeling of DNA, yellow, histones, and magenta, aptamer KU7–647 (bottom, right). White areas in the merged image represent close proximity of DNA, histone and aptamer. Representative images, captured with 40× oil and 2.8× zoom. Scale is equivalent to 10 µm. b Aptamer inhibition of NETs-mediated cytotoxicity of endothelial cells determined by MTS assay. EA.hy926 cells treated with 8 µg per well of NETs material (based on DNA concentration) and/or 8 µg per well (10.66 µM) of aptamer (KU7); * p

    Techniques Used: Derivative Assay, Confocal Microscopy, Labeling, Inhibition, MTS Assay, Concentration Assay

    Identification of histone-specific RNA aptamers using SELEX. a ). b Binding of Round 0 (R0) and Round 8 (R8) RNA to recombinant human histone H3 (top, left panel) and H4 (top, right panel) proteins. Binding of R8 RNA to human albumin (bottom, left panel) and human serum (bottom, right panel). c Percent sequence enrichment (% Enrichment) at each round of selection (black circle). The 50% sequence enrichment point (gray circle) is indicated for each selection
    Figure Legend Snippet: Identification of histone-specific RNA aptamers using SELEX. a ). b Binding of Round 0 (R0) and Round 8 (R8) RNA to recombinant human histone H3 (top, left panel) and H4 (top, right panel) proteins. Binding of R8 RNA to human albumin (bottom, left panel) and human serum (bottom, right panel). c Percent sequence enrichment (% Enrichment) at each round of selection (black circle). The 50% sequence enrichment point (gray circle) is indicated for each selection

    Techniques Used: Binding Assay, Recombinant, Sequencing, Selection

    Binding characterization and stability measurements of individual histone RNA aptamer sequences. a Binding kinetic rate constants ( k a and K D ) determined for aptamers KU7 (left panels) and KU9 (right panels) binding to CTH (top panels), H4 (middle panels) and BSA (bottom panels). Aptamers concentrations tested: 100 nM (blue), 50 nM (black), 25 nM (red), 12.5 nM (green), 10 nM (magenta). b Serum stability measurements for aptamers KU7 and KU9 (5 μM) in 50% human serum. T 1/2 KU7 = 150 h. T 1/2 KU9 = 48 h
    Figure Legend Snippet: Binding characterization and stability measurements of individual histone RNA aptamer sequences. a Binding kinetic rate constants ( k a and K D ) determined for aptamers KU7 (left panels) and KU9 (right panels) binding to CTH (top panels), H4 (middle panels) and BSA (bottom panels). Aptamers concentrations tested: 100 nM (blue), 50 nM (black), 25 nM (red), 12.5 nM (green), 10 nM (magenta). b Serum stability measurements for aptamers KU7 and KU9 (5 μM) in 50% human serum. T 1/2 KU7 = 150 h. T 1/2 KU9 = 48 h

    Techniques Used: Binding Assay

    Efficacy of histone aptamer in murine model of MODS. a Survival curves of mice injected IV with CTH in the presence or absence of aptamer treatment; n = 6 per group. Molar ratio of CTH to aptamer indicated. b Weights of the liver, lung and spleen normalized to pre-treatment body weight; * p
    Figure Legend Snippet: Efficacy of histone aptamer in murine model of MODS. a Survival curves of mice injected IV with CTH in the presence or absence of aptamer treatment; n = 6 per group. Molar ratio of CTH to aptamer indicated. b Weights of the liver, lung and spleen normalized to pre-treatment body weight; * p

    Techniques Used: Mouse Assay, Injection

    In vitro efficacy of RNA aptamers. a Human platelet aggregation measurements using platelets derived from three independent healthy donors. Collagen (Col), histone H4 (H4), histone aptamers (KU7 and KU9), calf thymus histones (CTH), heparin (Hep); * p
    Figure Legend Snippet: In vitro efficacy of RNA aptamers. a Human platelet aggregation measurements using platelets derived from three independent healthy donors. Collagen (Col), histone H4 (H4), histone aptamers (KU7 and KU9), calf thymus histones (CTH), heparin (Hep); * p

    Techniques Used: In Vitro, Derivative Assay

    5) Product Images from "Recombinant Thrombomodulin Protects Mice against Histone-Induced Lethal Thromboembolism"

    Article Title: Recombinant Thrombomodulin Protects Mice against Histone-Induced Lethal Thromboembolism

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0075961

    Recombinant thrombomodulin (rTM) protects mice against histone-induced fatal thrombosis. (A) The protective effect of rTM on histone-induced thrombocytopenia. Pretreatment with rTM (40 or 80 µg/g) 30 min before histone injection (40 µg/g) prevented histone-induced thrombocytopenia in mice (n = 3-4 per group, mean ± S.D.). ** P
    Figure Legend Snippet: Recombinant thrombomodulin (rTM) protects mice against histone-induced fatal thrombosis. (A) The protective effect of rTM on histone-induced thrombocytopenia. Pretreatment with rTM (40 or 80 µg/g) 30 min before histone injection (40 µg/g) prevented histone-induced thrombocytopenia in mice (n = 3-4 per group, mean ± S.D.). ** P

    Techniques Used: Recombinant, Mouse Assay, Injection

    Recombinant thrombomodulin (rTM) suppresses the activity of extracellular histones. (A) Binding assays with a quartz crystal microbalance (QCM) twin sensor system. Two channels of a sensor chip were coated with either histones (1 mg/ml) or BSA (1 mg/ml). The sensor chip was placed into the NAPiCOS Auto and then perfused with rTM (1 mg/ml). The interaction between molecules was recognized as the change in frequency of a quartz crystal resonator. (B) Inhibition of histone H4-mediated platelet aggregation by rTM. Washed platelets were stimulated with histone H4 (5 µg/ml) preincubated with rTM (5-25 µg/ml) or BSA (25 µg/ml). rTM, but not BSA, inhibited histone-induced platelet aggregation (n = 3-7 per group, mean ± S.D.). (C) Inhibition of histone H3-induced platelet aggregation by rTM. Washed platelets were stimulated with histone H3 (25 µg/ml) or collagen (1.44 µg/ml) in the presence or absence of rTM (15 µg/ml). rTM inhibited histone-induced platelet aggregation, but not collagen-induced platelet aggregation. Representative data of three independent experiments are shown. ** P
    Figure Legend Snippet: Recombinant thrombomodulin (rTM) suppresses the activity of extracellular histones. (A) Binding assays with a quartz crystal microbalance (QCM) twin sensor system. Two channels of a sensor chip were coated with either histones (1 mg/ml) or BSA (1 mg/ml). The sensor chip was placed into the NAPiCOS Auto and then perfused with rTM (1 mg/ml). The interaction between molecules was recognized as the change in frequency of a quartz crystal resonator. (B) Inhibition of histone H4-mediated platelet aggregation by rTM. Washed platelets were stimulated with histone H4 (5 µg/ml) preincubated with rTM (5-25 µg/ml) or BSA (25 µg/ml). rTM, but not BSA, inhibited histone-induced platelet aggregation (n = 3-7 per group, mean ± S.D.). (C) Inhibition of histone H3-induced platelet aggregation by rTM. Washed platelets were stimulated with histone H3 (25 µg/ml) or collagen (1.44 µg/ml) in the presence or absence of rTM (15 µg/ml). rTM inhibited histone-induced platelet aggregation, but not collagen-induced platelet aggregation. Representative data of three independent experiments are shown. ** P

    Techniques Used: Recombinant, Activity Assay, Binding Assay, Chromatin Immunoprecipitation, Inhibition

    Extracellular histones cause consumptive coagulopathy. Plasma fibrinogen (A), APTT (B), and PT (C) of mice 10 minutes after injection with vehicle or histones (20-80 µg/g, n = 4-10). ** P
    Figure Legend Snippet: Extracellular histones cause consumptive coagulopathy. Plasma fibrinogen (A), APTT (B), and PT (C) of mice 10 minutes after injection with vehicle or histones (20-80 µg/g, n = 4-10). ** P

    Techniques Used: Mouse Assay, Injection

    Extracellular histones cause fatal thromboembolism in mice. (A) The lethal effect of extracellular histones. Mice were intravenously injected with histones (0-80 µg/g, n = 7-12 per group), and survival was analyzed. (B) Histone-induced thrombocytopenia. Numbers of platelets (PLT), red blood cells (RBC), and white blood cells (WBC) in blood 10 min after infusion with histones (0-95 µg/g, n = 3-7 per group, mean ± S.D.) are shown. Data are presented as percentage of the vehicle group (0 µg/g histones). (C) Distribution of DyLight488-labeled platelets and Alexa-Fluor 594-labeled fibrin(ogen) in lung tissue 10 min after infusion with vehicle or 75 µg/g histones. Nuclei were stained with DAPI. Representative images of n = 4. Scale bar = 100 µm. * P
    Figure Legend Snippet: Extracellular histones cause fatal thromboembolism in mice. (A) The lethal effect of extracellular histones. Mice were intravenously injected with histones (0-80 µg/g, n = 7-12 per group), and survival was analyzed. (B) Histone-induced thrombocytopenia. Numbers of platelets (PLT), red blood cells (RBC), and white blood cells (WBC) in blood 10 min after infusion with histones (0-95 µg/g, n = 3-7 per group, mean ± S.D.) are shown. Data are presented as percentage of the vehicle group (0 µg/g histones). (C) Distribution of DyLight488-labeled platelets and Alexa-Fluor 594-labeled fibrin(ogen) in lung tissue 10 min after infusion with vehicle or 75 µg/g histones. Nuclei were stained with DAPI. Representative images of n = 4. Scale bar = 100 µm. * P

    Techniques Used: Mouse Assay, Injection, Labeling, Staining

    Extracellular histones cause acute right-sided heart failure and ventricular arrest. (A) ECG in mice intravenously injected with vehicle or histones (80 µg/g). The voltage between the right limb and the feet (lead II) was recorded. Data presented are representative of three independent experiments. (B) Still images from Movies S1-S3. Transthoracic echocardiography was performed in mice before and after intravenous injection of histones. Extracellular histones caused dilatation of the right ventricle (RV) and displacement of the interventricular septum toward the left ventricle (LV).
    Figure Legend Snippet: Extracellular histones cause acute right-sided heart failure and ventricular arrest. (A) ECG in mice intravenously injected with vehicle or histones (80 µg/g). The voltage between the right limb and the feet (lead II) was recorded. Data presented are representative of three independent experiments. (B) Still images from Movies S1-S3. Transthoracic echocardiography was performed in mice before and after intravenous injection of histones. Extracellular histones caused dilatation of the right ventricle (RV) and displacement of the interventricular septum toward the left ventricle (LV).

    Techniques Used: Mouse Assay, Injection

    Histone H3 levels, as measured by ELISA, in plasma of patients with sepsis and DIC. Plasma histone H3 levels were significantly higher in non-survivors (n = 16, minimum = 0, maximum = 1464.4, median = 15.5) compared with healthy volunteers (n = 15, minimum = 0, maximum = 0) as analyzed by Steel-Dwass test.
    Figure Legend Snippet: Histone H3 levels, as measured by ELISA, in plasma of patients with sepsis and DIC. Plasma histone H3 levels were significantly higher in non-survivors (n = 16, minimum = 0, maximum = 1464.4, median = 15.5) compared with healthy volunteers (n = 15, minimum = 0, maximum = 0) as analyzed by Steel-Dwass test.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    6) Product Images from "Notch- and Transducin-like Enhancer of Split (TLE)-dependent Histone Deacetylation Explain Interleukin 12 (IL-12) p70 Inhibition by Zymosan"

    Article Title: Notch- and Transducin-like Enhancer of Split (TLE)-dependent Histone Deacetylation Explain Interleukin 12 (IL-12) p70 Inhibition by Zymosan

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.222158

    Consensus sequences recognized by PKA and MSK and phosphorylation of c-Rel RHD and histone H3.3. Consensus sequences recognized by PKA and MSK in RelA/p65, c-Rel, and histone H3.3 are shown in A. In vitro phosphorylation of c-Rel RHD and the S267A mutant
    Figure Legend Snippet: Consensus sequences recognized by PKA and MSK and phosphorylation of c-Rel RHD and histone H3.3. Consensus sequences recognized by PKA and MSK in RelA/p65, c-Rel, and histone H3.3 are shown in A. In vitro phosphorylation of c-Rel RHD and the S267A mutant

    Techniques Used: In Vitro, Mutagenesis

    7) Product Images from "A functional SUMO-motif in the active site of PIM1 promotes its degradation via RNF4, and stimulates protein kinase activity"

    Article Title: A functional SUMO-motif in the active site of PIM1 promotes its degradation via RNF4, and stimulates protein kinase activity

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-03775-w

    SUMOylation increase PIM1 kinase activity in vitro . ( a ) Bacterially purified 6His-PIM1 was SUMOylated in vitro using purified GST-SUMO2. Equal amounts of SUMOylated protein (including PIM1) were captured using GST-beads and incubated without or with SENP1 catalytic domain for 1 hour at 30 °C. Kinase assays were then performed using Histone H3.3 as a substrate for at 30 °C for 0, 15, 30 and 45 min. Kinase activity of SUMO2-modified or unmodified PIM1 was measured by analyzing Histone H3.3 phosphorylation using a phospho-specific antibody. Equal levels of substrate and kinase were confirmed by western blotting using indicated antibodies. ( b ) Purified WT PIM1 was first incubated with or without SENP1 catalytic domain fragment for 1 hour at 30 °C, and immediately used in a kinase assay using Histone H3.3 as substrate for 30 min at 30 °C. PIM1 kinase activity was measured by analyzing Histone H3.3 phosphorylation using a phospho-specific antibody. Equal levels of substrate and kinase were confirmed by coomassie staining of the gel.
    Figure Legend Snippet: SUMOylation increase PIM1 kinase activity in vitro . ( a ) Bacterially purified 6His-PIM1 was SUMOylated in vitro using purified GST-SUMO2. Equal amounts of SUMOylated protein (including PIM1) were captured using GST-beads and incubated without or with SENP1 catalytic domain for 1 hour at 30 °C. Kinase assays were then performed using Histone H3.3 as a substrate for at 30 °C for 0, 15, 30 and 45 min. Kinase activity of SUMO2-modified or unmodified PIM1 was measured by analyzing Histone H3.3 phosphorylation using a phospho-specific antibody. Equal levels of substrate and kinase were confirmed by western blotting using indicated antibodies. ( b ) Purified WT PIM1 was first incubated with or without SENP1 catalytic domain fragment for 1 hour at 30 °C, and immediately used in a kinase assay using Histone H3.3 as substrate for 30 min at 30 °C. PIM1 kinase activity was measured by analyzing Histone H3.3 phosphorylation using a phospho-specific antibody. Equal levels of substrate and kinase were confirmed by coomassie staining of the gel.

    Techniques Used: Activity Assay, In Vitro, Purification, Incubation, Modification, Western Blot, Kinase Assay, Staining

    Identification of the sites of SUMOylation in PIM1. ( a ) Schematic showing the kinase domain of PIM1, and the position of various lysine (K) and and glutamic acid (E) residues predicted to be involved in SUMOylation. ( b ) WT PIM1 or single amino acid substitution site mutants were expressed at near equal protein levels in COS7 cells with 6His-SUMO2, by transfecting different amounts of plasmids. Empty vector was included, where appropriate, to maintain equal amounts of transfected plasmid DNA. An aliquot of whole cell lysate was taken as input, and the remainder subjected to Ni 2+ -NTA pull-down to capture SUMOylated proteins. The samples were subjected to SDS-PAGE followed by western blotting using a PIM1 antibody.
    Figure Legend Snippet: Identification of the sites of SUMOylation in PIM1. ( a ) Schematic showing the kinase domain of PIM1, and the position of various lysine (K) and and glutamic acid (E) residues predicted to be involved in SUMOylation. ( b ) WT PIM1 or single amino acid substitution site mutants were expressed at near equal protein levels in COS7 cells with 6His-SUMO2, by transfecting different amounts of plasmids. Empty vector was included, where appropriate, to maintain equal amounts of transfected plasmid DNA. An aliquot of whole cell lysate was taken as input, and the remainder subjected to Ni 2+ -NTA pull-down to capture SUMOylated proteins. The samples were subjected to SDS-PAGE followed by western blotting using a PIM1 antibody.

    Techniques Used: Plasmid Preparation, Transfection, SDS Page, Western Blot

    SUMOylation negatively regulates PIM1 protein levels by promoting its ubiquitylation and proteasomal degradation via RNF4. ( a ) H1299 or HeLa cells were transfected with siRNA targeting UBC9 (siUBC9), or non-targeting siRNA (NS) as negative control. Lysates were harvested at the indicated time points, and western blotting was performed for endogenous PIM1 (using 12H8 antibody), SUMO2 and UBC9. Actin was used as a loading control. ( b ) H1299 cells were transfected with a plasmid expressing 6His-PIM1 in the absence and presence of Ubc9 siRNA, and PIM1 was affinity purified under denaturing conditions as done previously for 6His-SUMO protein. Eluted proteins were analyzed by western blotting using SUMO2 antibody to detect SUMOylated PIM1. ( c ) SUMOylation assay was done in COS7 cells transfected with plasmids expressing MYC-tagged PIM1 alone or with HA-UBC9 and 6His-SUMO2 to stimulate PIM1 SUMOylation. Cells were additionally transfected with plasmids expressing WT RNF4 or RING finger mutant RNF4 (mRING) or SUMO-interaction motif mutant RNF4 (mSIM). PIM1 SUMOylation was analyzed by western blotting of Ni 2+ -NTA pull-down samples using PIM1 (12H8) antibody. Western blotting was also performed on the input samples using indicated antibodies to check expression of transfected proteins. ( d ) H1299 cells were transfected with siRNA targeting RNF4, PIM1, or non-targeting siRNA (NS) as negative control for the indicated time points. Western blotting was performed for endogenous PIM1 (using 12H8 antibody) and RNF4. Actin was used as a loading control. ( e ) Ubiquitylation assay was performed in H1299 cells transfected with plasmids expressing MYC-tagged WT PIM1, K169R, E171A and K67M with 6His-ubiquitin. Ubiquitylated PIM1 was detected by western blotting using 12H8 (PIM1) antibody. ( f ) Ubiquitylation assay was performed in parental H1299 cells, or H1299 RNF4 knock-out cells, transfected with plasmids expressing MYC-tagged WT PIM1 and 6His-ubiquitin. Ubiquitylated PIM1 was detected by western blotting using PIM1 (12H8) antibody. ( g ) Ubiquitylation assay was performed in H1299 cells transected with plasmids expressing MYC-tagged WT PIM1 and Flag-tagged PIAS3, with or without 6His-ubiquitin. Ubiquitylated PIM1 was detected by western blotting using PIM1 (12H8) antibody.
    Figure Legend Snippet: SUMOylation negatively regulates PIM1 protein levels by promoting its ubiquitylation and proteasomal degradation via RNF4. ( a ) H1299 or HeLa cells were transfected with siRNA targeting UBC9 (siUBC9), or non-targeting siRNA (NS) as negative control. Lysates were harvested at the indicated time points, and western blotting was performed for endogenous PIM1 (using 12H8 antibody), SUMO2 and UBC9. Actin was used as a loading control. ( b ) H1299 cells were transfected with a plasmid expressing 6His-PIM1 in the absence and presence of Ubc9 siRNA, and PIM1 was affinity purified under denaturing conditions as done previously for 6His-SUMO protein. Eluted proteins were analyzed by western blotting using SUMO2 antibody to detect SUMOylated PIM1. ( c ) SUMOylation assay was done in COS7 cells transfected with plasmids expressing MYC-tagged PIM1 alone or with HA-UBC9 and 6His-SUMO2 to stimulate PIM1 SUMOylation. Cells were additionally transfected with plasmids expressing WT RNF4 or RING finger mutant RNF4 (mRING) or SUMO-interaction motif mutant RNF4 (mSIM). PIM1 SUMOylation was analyzed by western blotting of Ni 2+ -NTA pull-down samples using PIM1 (12H8) antibody. Western blotting was also performed on the input samples using indicated antibodies to check expression of transfected proteins. ( d ) H1299 cells were transfected with siRNA targeting RNF4, PIM1, or non-targeting siRNA (NS) as negative control for the indicated time points. Western blotting was performed for endogenous PIM1 (using 12H8 antibody) and RNF4. Actin was used as a loading control. ( e ) Ubiquitylation assay was performed in H1299 cells transfected with plasmids expressing MYC-tagged WT PIM1, K169R, E171A and K67M with 6His-ubiquitin. Ubiquitylated PIM1 was detected by western blotting using 12H8 (PIM1) antibody. ( f ) Ubiquitylation assay was performed in parental H1299 cells, or H1299 RNF4 knock-out cells, transfected with plasmids expressing MYC-tagged WT PIM1 and 6His-ubiquitin. Ubiquitylated PIM1 was detected by western blotting using PIM1 (12H8) antibody. ( g ) Ubiquitylation assay was performed in H1299 cells transected with plasmids expressing MYC-tagged WT PIM1 and Flag-tagged PIAS3, with or without 6His-ubiquitin. Ubiquitylated PIM1 was detected by western blotting using PIM1 (12H8) antibody.

    Techniques Used: Transfection, Negative Control, Western Blot, Plasmid Preparation, Expressing, Affinity Purification, Mutagenesis, Ubiquitin Assay, Knock-Out

    Model for regulation of PIM1 by SUMOylation. The bi-lobed structure of PIM1 kinase is shown in blue. The consensus SUMOylated lysine residue is located in the substrate binding pocket (K169 in deep blue). For the purpose of presentation, the non-consensus lysine is shown in blue in the N-terminal domain. Stimuli such as growth factors or stress might induce SUMOylation of PIM1 under endogenous conditions. SUMOylated PIM1 can bind and phosphorylate substrates. Once this is achieved, a SUMO targeted ubiquitin ligase is recruited to polySUMOylated PIM1 leading to attachment of polyubiquitin chains on PIM1. The SUMOylated and ubiquitylated PIM1 is then targeted for degradation by the proteasome.
    Figure Legend Snippet: Model for regulation of PIM1 by SUMOylation. The bi-lobed structure of PIM1 kinase is shown in blue. The consensus SUMOylated lysine residue is located in the substrate binding pocket (K169 in deep blue). For the purpose of presentation, the non-consensus lysine is shown in blue in the N-terminal domain. Stimuli such as growth factors or stress might induce SUMOylation of PIM1 under endogenous conditions. SUMOylated PIM1 can bind and phosphorylate substrates. Once this is achieved, a SUMO targeted ubiquitin ligase is recruited to polySUMOylated PIM1 leading to attachment of polyubiquitin chains on PIM1. The SUMOylated and ubiquitylated PIM1 is then targeted for degradation by the proteasome.

    Techniques Used: Binding Assay

    PIAS family members can directly interact with PIM1, and act as E3 SUMO ligases for PIM1. ( a ) Western blots showing SUMOylation assay performed in COS7 cells transfected with plasmids expressing PIM1, 6His-SUMO2 with PIAS1, PIAS3 or PIASy in the absence or presence of MG132 (20 μM for 6 hours). A western blot of whole cell lysate (input) was also performed to confirm the expression PIM1, PIAS1, PIAS3 and PIAS3 using the indicated antibodies. ( b ) H1299 cells were co-transfected with PIM1 and PIAS3 expression plasmids, and co-immunoprecipitation was performed using anti-Flag-antibody to pull-down PIAS3 associated complexes. The immunoprecipitated (IP) samples were analyzed by western blotting using Flag-tag and PIM1 (12H8) antibodies. Mouse IgG was used as a negative control. ( c ) H1299 cells were transfected with plasmids expressing PIM1 and PIAS1 in the presence or absence of MG132 (20 μM for 6 hours), and co-immunoprecipitation was performed using HA-tag (PIAS1) antibody. The IP samples were western blotted for the presence of PIAS1 and PIM1 using anti-HA- and PIM1 (12H8) antibodies. Mouse IgG was used as a negative control. ( d ) H1299 cells were co-transfected with PIM1 and PIASy expression plasmids, and co-immunoprecipitation was performed using anti-HA-antibody to pull-down PIASy associated complexes. The immunoprecipitated (IP) samples were analyzed by western blotting using anti-HA (PIASy) and PIM1 (12H8) antibodies. Mouse IgG was used as a negative control.
    Figure Legend Snippet: PIAS family members can directly interact with PIM1, and act as E3 SUMO ligases for PIM1. ( a ) Western blots showing SUMOylation assay performed in COS7 cells transfected with plasmids expressing PIM1, 6His-SUMO2 with PIAS1, PIAS3 or PIASy in the absence or presence of MG132 (20 μM for 6 hours). A western blot of whole cell lysate (input) was also performed to confirm the expression PIM1, PIAS1, PIAS3 and PIAS3 using the indicated antibodies. ( b ) H1299 cells were co-transfected with PIM1 and PIAS3 expression plasmids, and co-immunoprecipitation was performed using anti-Flag-antibody to pull-down PIAS3 associated complexes. The immunoprecipitated (IP) samples were analyzed by western blotting using Flag-tag and PIM1 (12H8) antibodies. Mouse IgG was used as a negative control. ( c ) H1299 cells were transfected with plasmids expressing PIM1 and PIAS1 in the presence or absence of MG132 (20 μM for 6 hours), and co-immunoprecipitation was performed using HA-tag (PIAS1) antibody. The IP samples were western blotted for the presence of PIAS1 and PIM1 using anti-HA- and PIM1 (12H8) antibodies. Mouse IgG was used as a negative control. ( d ) H1299 cells were co-transfected with PIM1 and PIASy expression plasmids, and co-immunoprecipitation was performed using anti-HA-antibody to pull-down PIASy associated complexes. The immunoprecipitated (IP) samples were analyzed by western blotting using anti-HA (PIASy) and PIM1 (12H8) antibodies. Mouse IgG was used as a negative control.

    Techniques Used: Activated Clotting Time Assay, Western Blot, Transfection, Expressing, Immunoprecipitation, FLAG-tag, Negative Control

    PIM1 SUMOylation regulates substrate specificity in vitro and in cultured cells. ( a ) 6His-PIM1 (WT or mutant) was expressed and purified from bacterial cells, and resolved by SDS-PAGE. A western blot for the same samples was also performed using a pan-phospho tyrosine antibody to detect PIM1 autophosphorylation. ( b ) The purified 6His-PIM1 proteins were treated with lambda phosphatase (+) to remove overall phosphorylation or untreated (−). Samples were resolved by SDS-PAGE, and stained with coomassie to visualize a shift in mobility, which is indicative of dephosphorylation. ( c ) In vitro kinase assays were carried out using recombinant c-MYC or Histone H3.3 as substrates, in the absence or presence of the indicated purified 6His-PIM1 proteins. The samples were resolved by SDS-PAGE, and either stained with coomassie to detect total protein levels or transferred to a nitrocellulose membrane for western blotting using phospho-specific antibodies as a measure of PIM1 kinase activity. ( d ) U2OS-FRT cells expressing YFP alone, YFP-WT PIM1 and YFP-E171A were treated with 10 ng/ml doxycycline; U2OS-FRT expressing YFP-K169R was treated with 20 ng/ml doxycycline and U2OS-FRT expressing YFP-K67M was treated with 50 ng/ml doxycycline for 48 hours, followed by western blotting using indicated antibodies.
    Figure Legend Snippet: PIM1 SUMOylation regulates substrate specificity in vitro and in cultured cells. ( a ) 6His-PIM1 (WT or mutant) was expressed and purified from bacterial cells, and resolved by SDS-PAGE. A western blot for the same samples was also performed using a pan-phospho tyrosine antibody to detect PIM1 autophosphorylation. ( b ) The purified 6His-PIM1 proteins were treated with lambda phosphatase (+) to remove overall phosphorylation or untreated (−). Samples were resolved by SDS-PAGE, and stained with coomassie to visualize a shift in mobility, which is indicative of dephosphorylation. ( c ) In vitro kinase assays were carried out using recombinant c-MYC or Histone H3.3 as substrates, in the absence or presence of the indicated purified 6His-PIM1 proteins. The samples were resolved by SDS-PAGE, and either stained with coomassie to detect total protein levels or transferred to a nitrocellulose membrane for western blotting using phospho-specific antibodies as a measure of PIM1 kinase activity. ( d ) U2OS-FRT cells expressing YFP alone, YFP-WT PIM1 and YFP-E171A were treated with 10 ng/ml doxycycline; U2OS-FRT expressing YFP-K169R was treated with 20 ng/ml doxycycline and U2OS-FRT expressing YFP-K67M was treated with 50 ng/ml doxycycline for 48 hours, followed by western blotting using indicated antibodies.

    Techniques Used: In Vitro, Cell Culture, Mutagenesis, Purification, SDS Page, Western Blot, Staining, De-Phosphorylation Assay, Recombinant, Activity Assay, Expressing

    SUMOylation of PIM1 in vitro and in cultured cells. ( a ) In vitro transcribed and translated 35 S-methionine labeled PIM1 was incubated with recombinant SAE1/2, UBC9 with SUMO1 or SUMO2 in the presence of ATP-regeneration system. SP100 was used as a positive control in the SUMOylation reaction. SUMOylation of radiolabelled PIM1 was visualized on a Phosphorimager. ( b ) Bacterially expressed and purified GST-PIM1 was incubated in the presence of ATP, recombinant SAE1/2, UBC9 with SUMO1 (left) or SUMO2 (right). SUMOylated PIM1 was detected by western blotting using a GST-tag antibody. ( c ) COS7 cells were transfected with plasmids encoding MYC-tagged PIM1 alone or in combination with 6His-SUMO1, 2 or 3 and SUMOylation assay was carried out using Ni 2+ -NTA beads, 42–48 hours post transfection. PIM1 SUMOylation was analyzed by western blotting of Ni 2+ -NTA pull-down samples using MYC-tag (9E10) antibody. Total levels of PIM1 expressed under each transfection condition were analyzed by western blotting of Input samples using MYC-tag (9E10) antibody. ( d ) H1299 cells were transfected with a plasmid expressing 6His-PIM1, and PIM1 was affinity purified under denaturing conditions as done previously for 6His-SUMO protein. Eluted proteins were analyzed by western blotting using PIM1 (12H8) and SUMO2 antibody to detect SUMOylated PIM1. ( e ) COS7 cells were transfected with plasmids expressing PIM1 alone, or with 6His-SUMO2 in combination with catalytically active Flag-SENP1 (WT) or inactive Flag-SENP1 (MT), and SUMOylation assay was performed to isolate SUMOylated proteins. PIM1 SUMOylation was analyzed by western blotting using PIM1 (12H8) antibody. Western blotting of whole cell lysate or input was done using Flag-tag antibody to confirm expression of SENP1. Empty vector was included, where appropriate, to maintain equal amounts of transfected plasmid DNA. ( f ) Lysates from K562 cells (treated with 20 μM MG132 for 6 hours) were incubated at 30 °C for 30 minutes in the absence or presence of 50 nM recombinant catalytic domain of SUMO protease, SENP1, followed by western blotting using PIM1 (12H8) antibody.
    Figure Legend Snippet: SUMOylation of PIM1 in vitro and in cultured cells. ( a ) In vitro transcribed and translated 35 S-methionine labeled PIM1 was incubated with recombinant SAE1/2, UBC9 with SUMO1 or SUMO2 in the presence of ATP-regeneration system. SP100 was used as a positive control in the SUMOylation reaction. SUMOylation of radiolabelled PIM1 was visualized on a Phosphorimager. ( b ) Bacterially expressed and purified GST-PIM1 was incubated in the presence of ATP, recombinant SAE1/2, UBC9 with SUMO1 (left) or SUMO2 (right). SUMOylated PIM1 was detected by western blotting using a GST-tag antibody. ( c ) COS7 cells were transfected with plasmids encoding MYC-tagged PIM1 alone or in combination with 6His-SUMO1, 2 or 3 and SUMOylation assay was carried out using Ni 2+ -NTA beads, 42–48 hours post transfection. PIM1 SUMOylation was analyzed by western blotting of Ni 2+ -NTA pull-down samples using MYC-tag (9E10) antibody. Total levels of PIM1 expressed under each transfection condition were analyzed by western blotting of Input samples using MYC-tag (9E10) antibody. ( d ) H1299 cells were transfected with a plasmid expressing 6His-PIM1, and PIM1 was affinity purified under denaturing conditions as done previously for 6His-SUMO protein. Eluted proteins were analyzed by western blotting using PIM1 (12H8) and SUMO2 antibody to detect SUMOylated PIM1. ( e ) COS7 cells were transfected with plasmids expressing PIM1 alone, or with 6His-SUMO2 in combination with catalytically active Flag-SENP1 (WT) or inactive Flag-SENP1 (MT), and SUMOylation assay was performed to isolate SUMOylated proteins. PIM1 SUMOylation was analyzed by western blotting using PIM1 (12H8) antibody. Western blotting of whole cell lysate or input was done using Flag-tag antibody to confirm expression of SENP1. Empty vector was included, where appropriate, to maintain equal amounts of transfected plasmid DNA. ( f ) Lysates from K562 cells (treated with 20 μM MG132 for 6 hours) were incubated at 30 °C for 30 minutes in the absence or presence of 50 nM recombinant catalytic domain of SUMO protease, SENP1, followed by western blotting using PIM1 (12H8) antibody.

    Techniques Used: In Vitro, Cell Culture, Labeling, Incubation, Recombinant, Positive Control, Purification, Western Blot, Transfection, Plasmid Preparation, Expressing, Affinity Purification, FLAG-tag

    SUMOylation regulates PIM1 stability. ( a ) Plasmids encoding WT MYC-PIM1, PIM1 SUMO mutants (MYC-K169R and MYC-E171A) and a catalytically inactive mutant (MYC-K67M) were transiently transfected into H1299 cells. Cycloheximide was added to the cells 24 hours post transfection at a final concentration of 50 μg/ml to inhibit protein synthesis, and harvested at the indicated time points. The cell lysates were analyzed by western blotting using PIM1 (12H8) antibody. Actin was used as a loading control. ( b ) PIM1 band intensity in each case was quantified relative to the zero time point, using Biorad ImageLab software, and plotted on a graph as percentage of protein remaining in log scale. ( c ) HeLa-FRT cells expressing YFP-tagged WT PIM1 or mutant PIM1 were treated with 50 ng/ml doxycycline for 24 hours to induce protein expression, following which cycloheximide chase assay was performed as done in panel A. ( d ) PIM1 protein levels were quantified and represented graphically as done in panel B.
    Figure Legend Snippet: SUMOylation regulates PIM1 stability. ( a ) Plasmids encoding WT MYC-PIM1, PIM1 SUMO mutants (MYC-K169R and MYC-E171A) and a catalytically inactive mutant (MYC-K67M) were transiently transfected into H1299 cells. Cycloheximide was added to the cells 24 hours post transfection at a final concentration of 50 μg/ml to inhibit protein synthesis, and harvested at the indicated time points. The cell lysates were analyzed by western blotting using PIM1 (12H8) antibody. Actin was used as a loading control. ( b ) PIM1 band intensity in each case was quantified relative to the zero time point, using Biorad ImageLab software, and plotted on a graph as percentage of protein remaining in log scale. ( c ) HeLa-FRT cells expressing YFP-tagged WT PIM1 or mutant PIM1 were treated with 50 ng/ml doxycycline for 24 hours to induce protein expression, following which cycloheximide chase assay was performed as done in panel A. ( d ) PIM1 protein levels were quantified and represented graphically as done in panel B.

    Techniques Used: Mutagenesis, Transfection, Concentration Assay, Western Blot, Software, Expressing

    8) Product Images from "A functional SUMO-motif in the active site of PIM1 promotes its degradation via RNF4, and stimulates protein kinase activity"

    Article Title: A functional SUMO-motif in the active site of PIM1 promotes its degradation via RNF4, and stimulates protein kinase activity

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-03775-w

    SUMOylation increase PIM1 kinase activity in vitro . ( a ) Bacterially purified 6His-PIM1 was SUMOylated in vitro using purified GST-SUMO2. Equal amounts of SUMOylated protein (including PIM1) were captured using GST-beads and incubated without or with SENP1 catalytic domain for 1 hour at 30 °C. Kinase assays were then performed using Histone H3.3 as a substrate for at 30 °C for 0, 15, 30 and 45 min. Kinase activity of SUMO2-modified or unmodified PIM1 was measured by analyzing Histone H3.3 phosphorylation using a phospho-specific antibody. Equal levels of substrate and kinase were confirmed by western blotting using indicated antibodies. ( b ) Purified WT PIM1 was first incubated with or without SENP1 catalytic domain fragment for 1 hour at 30 °C, and immediately used in a kinase assay using Histone H3.3 as substrate for 30 min at 30 °C. PIM1 kinase activity was measured by analyzing Histone H3.3 phosphorylation using a phospho-specific antibody. Equal levels of substrate and kinase were confirmed by coomassie staining of the gel.
    Figure Legend Snippet: SUMOylation increase PIM1 kinase activity in vitro . ( a ) Bacterially purified 6His-PIM1 was SUMOylated in vitro using purified GST-SUMO2. Equal amounts of SUMOylated protein (including PIM1) were captured using GST-beads and incubated without or with SENP1 catalytic domain for 1 hour at 30 °C. Kinase assays were then performed using Histone H3.3 as a substrate for at 30 °C for 0, 15, 30 and 45 min. Kinase activity of SUMO2-modified or unmodified PIM1 was measured by analyzing Histone H3.3 phosphorylation using a phospho-specific antibody. Equal levels of substrate and kinase were confirmed by western blotting using indicated antibodies. ( b ) Purified WT PIM1 was first incubated with or without SENP1 catalytic domain fragment for 1 hour at 30 °C, and immediately used in a kinase assay using Histone H3.3 as substrate for 30 min at 30 °C. PIM1 kinase activity was measured by analyzing Histone H3.3 phosphorylation using a phospho-specific antibody. Equal levels of substrate and kinase were confirmed by coomassie staining of the gel.

    Techniques Used: Activity Assay, In Vitro, Purification, Incubation, Modification, Western Blot, Kinase Assay, Staining

    Identification of the sites of SUMOylation in PIM1. ( a ) Schematic showing the kinase domain of PIM1, and the position of various lysine (K) and and glutamic acid (E) residues predicted to be involved in SUMOylation. ( b ) WT PIM1 or single amino acid substitution site mutants were expressed at near equal protein levels in COS7 cells with 6His-SUMO2, by transfecting different amounts of plasmids. Empty vector was included, where appropriate, to maintain equal amounts of transfected plasmid DNA. An aliquot of whole cell lysate was taken as input, and the remainder subjected to Ni 2+ -NTA pull-down to capture SUMOylated proteins. The samples were subjected to SDS-PAGE followed by western blotting using a PIM1 antibody.
    Figure Legend Snippet: Identification of the sites of SUMOylation in PIM1. ( a ) Schematic showing the kinase domain of PIM1, and the position of various lysine (K) and and glutamic acid (E) residues predicted to be involved in SUMOylation. ( b ) WT PIM1 or single amino acid substitution site mutants were expressed at near equal protein levels in COS7 cells with 6His-SUMO2, by transfecting different amounts of plasmids. Empty vector was included, where appropriate, to maintain equal amounts of transfected plasmid DNA. An aliquot of whole cell lysate was taken as input, and the remainder subjected to Ni 2+ -NTA pull-down to capture SUMOylated proteins. The samples were subjected to SDS-PAGE followed by western blotting using a PIM1 antibody.

    Techniques Used: Plasmid Preparation, Transfection, SDS Page, Western Blot

    SUMOylation negatively regulates PIM1 protein levels by promoting its ubiquitylation and proteasomal degradation via RNF4. ( a ) H1299 or HeLa cells were transfected with siRNA targeting UBC9 (siUBC9), or non-targeting siRNA (NS) as negative control. Lysates were harvested at the indicated time points, and western blotting was performed for endogenous PIM1 (using 12H8 antibody), SUMO2 and UBC9. Actin was used as a loading control. ( b ) H1299 cells were transfected with a plasmid expressing 6His-PIM1 in the absence and presence of Ubc9 siRNA, and PIM1 was affinity purified under denaturing conditions as done previously for 6His-SUMO protein. Eluted proteins were analyzed by western blotting using SUMO2 antibody to detect SUMOylated PIM1. ( c ) SUMOylation assay was done in COS7 cells transfected with plasmids expressing MYC-tagged PIM1 alone or with HA-UBC9 and 6His-SUMO2 to stimulate PIM1 SUMOylation. Cells were additionally transfected with plasmids expressing WT RNF4 or RING finger mutant RNF4 (mRING) or SUMO-interaction motif mutant RNF4 (mSIM). PIM1 SUMOylation was analyzed by western blotting of Ni 2+ -NTA pull-down samples using PIM1 (12H8) antibody. Western blotting was also performed on the input samples using indicated antibodies to check expression of transfected proteins. ( d ) H1299 cells were transfected with siRNA targeting RNF4, PIM1, or non-targeting siRNA (NS) as negative control for the indicated time points. Western blotting was performed for endogenous PIM1 (using 12H8 antibody) and RNF4. Actin was used as a loading control. ( e ) Ubiquitylation assay was performed in H1299 cells transfected with plasmids expressing MYC-tagged WT PIM1, K169R, E171A and K67M with 6His-ubiquitin. Ubiquitylated PIM1 was detected by western blotting using 12H8 (PIM1) antibody. ( f ) Ubiquitylation assay was performed in parental H1299 cells, or H1299 RNF4 knock-out cells, transfected with plasmids expressing MYC-tagged WT PIM1 and 6His-ubiquitin. Ubiquitylated PIM1 was detected by western blotting using PIM1 (12H8) antibody. ( g ) Ubiquitylation assay was performed in H1299 cells transected with plasmids expressing MYC-tagged WT PIM1 and Flag-tagged PIAS3, with or without 6His-ubiquitin. Ubiquitylated PIM1 was detected by western blotting using PIM1 (12H8) antibody.
    Figure Legend Snippet: SUMOylation negatively regulates PIM1 protein levels by promoting its ubiquitylation and proteasomal degradation via RNF4. ( a ) H1299 or HeLa cells were transfected with siRNA targeting UBC9 (siUBC9), or non-targeting siRNA (NS) as negative control. Lysates were harvested at the indicated time points, and western blotting was performed for endogenous PIM1 (using 12H8 antibody), SUMO2 and UBC9. Actin was used as a loading control. ( b ) H1299 cells were transfected with a plasmid expressing 6His-PIM1 in the absence and presence of Ubc9 siRNA, and PIM1 was affinity purified under denaturing conditions as done previously for 6His-SUMO protein. Eluted proteins were analyzed by western blotting using SUMO2 antibody to detect SUMOylated PIM1. ( c ) SUMOylation assay was done in COS7 cells transfected with plasmids expressing MYC-tagged PIM1 alone or with HA-UBC9 and 6His-SUMO2 to stimulate PIM1 SUMOylation. Cells were additionally transfected with plasmids expressing WT RNF4 or RING finger mutant RNF4 (mRING) or SUMO-interaction motif mutant RNF4 (mSIM). PIM1 SUMOylation was analyzed by western blotting of Ni 2+ -NTA pull-down samples using PIM1 (12H8) antibody. Western blotting was also performed on the input samples using indicated antibodies to check expression of transfected proteins. ( d ) H1299 cells were transfected with siRNA targeting RNF4, PIM1, or non-targeting siRNA (NS) as negative control for the indicated time points. Western blotting was performed for endogenous PIM1 (using 12H8 antibody) and RNF4. Actin was used as a loading control. ( e ) Ubiquitylation assay was performed in H1299 cells transfected with plasmids expressing MYC-tagged WT PIM1, K169R, E171A and K67M with 6His-ubiquitin. Ubiquitylated PIM1 was detected by western blotting using 12H8 (PIM1) antibody. ( f ) Ubiquitylation assay was performed in parental H1299 cells, or H1299 RNF4 knock-out cells, transfected with plasmids expressing MYC-tagged WT PIM1 and 6His-ubiquitin. Ubiquitylated PIM1 was detected by western blotting using PIM1 (12H8) antibody. ( g ) Ubiquitylation assay was performed in H1299 cells transected with plasmids expressing MYC-tagged WT PIM1 and Flag-tagged PIAS3, with or without 6His-ubiquitin. Ubiquitylated PIM1 was detected by western blotting using PIM1 (12H8) antibody.

    Techniques Used: Transfection, Negative Control, Western Blot, Plasmid Preparation, Expressing, Affinity Purification, Mutagenesis, Ubiquitin Assay, Knock-Out

    PIAS family members can directly interact with PIM1, and act as E3 SUMO ligases for PIM1. ( a ) Western blots showing SUMOylation assay performed in COS7 cells transfected with plasmids expressing PIM1, 6His-SUMO2 with PIAS1, PIAS3 or PIASy in the absence or presence of MG132 (20 μM for 6 hours). A western blot of whole cell lysate (input) was also performed to confirm the expression PIM1, PIAS1, PIAS3 and PIAS3 using the indicated antibodies. ( b ) H1299 cells were co-transfected with PIM1 and PIAS3 expression plasmids, and co-immunoprecipitation was performed using anti-Flag-antibody to pull-down PIAS3 associated complexes. The immunoprecipitated (IP) samples were analyzed by western blotting using Flag-tag and PIM1 (12H8) antibodies. Mouse IgG was used as a negative control. ( c ) H1299 cells were transfected with plasmids expressing PIM1 and PIAS1 in the presence or absence of MG132 (20 μM for 6 hours), and co-immunoprecipitation was performed using HA-tag (PIAS1) antibody. The IP samples were western blotted for the presence of PIAS1 and PIM1 using anti-HA- and PIM1 (12H8) antibodies. Mouse IgG was used as a negative control. ( d ) H1299 cells were co-transfected with PIM1 and PIASy expression plasmids, and co-immunoprecipitation was performed using anti-HA-antibody to pull-down PIASy associated complexes. The immunoprecipitated (IP) samples were analyzed by western blotting using anti-HA (PIASy) and PIM1 (12H8) antibodies. Mouse IgG was used as a negative control.
    Figure Legend Snippet: PIAS family members can directly interact with PIM1, and act as E3 SUMO ligases for PIM1. ( a ) Western blots showing SUMOylation assay performed in COS7 cells transfected with plasmids expressing PIM1, 6His-SUMO2 with PIAS1, PIAS3 or PIASy in the absence or presence of MG132 (20 μM for 6 hours). A western blot of whole cell lysate (input) was also performed to confirm the expression PIM1, PIAS1, PIAS3 and PIAS3 using the indicated antibodies. ( b ) H1299 cells were co-transfected with PIM1 and PIAS3 expression plasmids, and co-immunoprecipitation was performed using anti-Flag-antibody to pull-down PIAS3 associated complexes. The immunoprecipitated (IP) samples were analyzed by western blotting using Flag-tag and PIM1 (12H8) antibodies. Mouse IgG was used as a negative control. ( c ) H1299 cells were transfected with plasmids expressing PIM1 and PIAS1 in the presence or absence of MG132 (20 μM for 6 hours), and co-immunoprecipitation was performed using HA-tag (PIAS1) antibody. The IP samples were western blotted for the presence of PIAS1 and PIM1 using anti-HA- and PIM1 (12H8) antibodies. Mouse IgG was used as a negative control. ( d ) H1299 cells were co-transfected with PIM1 and PIASy expression plasmids, and co-immunoprecipitation was performed using anti-HA-antibody to pull-down PIASy associated complexes. The immunoprecipitated (IP) samples were analyzed by western blotting using anti-HA (PIASy) and PIM1 (12H8) antibodies. Mouse IgG was used as a negative control.

    Techniques Used: Activated Clotting Time Assay, Western Blot, Transfection, Expressing, Immunoprecipitation, FLAG-tag, Negative Control

    PIM1 SUMOylation regulates substrate specificity in vitro and in cultured cells. ( a ) 6His-PIM1 (WT or mutant) was expressed and purified from bacterial cells, and resolved by SDS-PAGE. A western blot for the same samples was also performed using a pan-phospho tyrosine antibody to detect PIM1 autophosphorylation. ( b ) The purified 6His-PIM1 proteins were treated with lambda phosphatase (+) to remove overall phosphorylation or untreated (−). Samples were resolved by SDS-PAGE, and stained with coomassie to visualize a shift in mobility, which is indicative of dephosphorylation. ( c ) In vitro kinase assays were carried out using recombinant c-MYC or Histone H3.3 as substrates, in the absence or presence of the indicated purified 6His-PIM1 proteins. The samples were resolved by SDS-PAGE, and either stained with coomassie to detect total protein levels or transferred to a nitrocellulose membrane for western blotting using phospho-specific antibodies as a measure of PIM1 kinase activity. ( d ) U2OS-FRT cells expressing YFP alone, YFP-WT PIM1 and YFP-E171A were treated with 10 ng/ml doxycycline; U2OS-FRT expressing YFP-K169R was treated with 20 ng/ml doxycycline and U2OS-FRT expressing YFP-K67M was treated with 50 ng/ml doxycycline for 48 hours, followed by western blotting using indicated antibodies.
    Figure Legend Snippet: PIM1 SUMOylation regulates substrate specificity in vitro and in cultured cells. ( a ) 6His-PIM1 (WT or mutant) was expressed and purified from bacterial cells, and resolved by SDS-PAGE. A western blot for the same samples was also performed using a pan-phospho tyrosine antibody to detect PIM1 autophosphorylation. ( b ) The purified 6His-PIM1 proteins were treated with lambda phosphatase (+) to remove overall phosphorylation or untreated (−). Samples were resolved by SDS-PAGE, and stained with coomassie to visualize a shift in mobility, which is indicative of dephosphorylation. ( c ) In vitro kinase assays were carried out using recombinant c-MYC or Histone H3.3 as substrates, in the absence or presence of the indicated purified 6His-PIM1 proteins. The samples were resolved by SDS-PAGE, and either stained with coomassie to detect total protein levels or transferred to a nitrocellulose membrane for western blotting using phospho-specific antibodies as a measure of PIM1 kinase activity. ( d ) U2OS-FRT cells expressing YFP alone, YFP-WT PIM1 and YFP-E171A were treated with 10 ng/ml doxycycline; U2OS-FRT expressing YFP-K169R was treated with 20 ng/ml doxycycline and U2OS-FRT expressing YFP-K67M was treated with 50 ng/ml doxycycline for 48 hours, followed by western blotting using indicated antibodies.

    Techniques Used: In Vitro, Cell Culture, Mutagenesis, Purification, SDS Page, Western Blot, Staining, De-Phosphorylation Assay, Recombinant, Activity Assay, Expressing

    SUMOylation of PIM1 in vitro and in cultured cells. ( a ) In vitro transcribed and translated 35 S-methionine labeled PIM1 was incubated with recombinant SAE1/2, UBC9 with SUMO1 or SUMO2 in the presence of ATP-regeneration system. SP100 was used as a positive control in the SUMOylation reaction. SUMOylation of radiolabelled PIM1 was visualized on a Phosphorimager. ( b ) Bacterially expressed and purified GST-PIM1 was incubated in the presence of ATP, recombinant SAE1/2, UBC9 with SUMO1 (left) or SUMO2 (right). SUMOylated PIM1 was detected by western blotting using a GST-tag antibody. ( c ) COS7 cells were transfected with plasmids encoding MYC-tagged PIM1 alone or in combination with 6His-SUMO1, 2 or 3 and SUMOylation assay was carried out using Ni 2+ -NTA beads, 42–48 hours post transfection. PIM1 SUMOylation was analyzed by western blotting of Ni 2+ -NTA pull-down samples using MYC-tag (9E10) antibody. Total levels of PIM1 expressed under each transfection condition were analyzed by western blotting of Input samples using MYC-tag (9E10) antibody. ( d ) H1299 cells were transfected with a plasmid expressing 6His-PIM1, and PIM1 was affinity purified under denaturing conditions as done previously for 6His-SUMO protein. Eluted proteins were analyzed by western blotting using PIM1 (12H8) and SUMO2 antibody to detect SUMOylated PIM1. ( e ) COS7 cells were transfected with plasmids expressing PIM1 alone, or with 6His-SUMO2 in combination with catalytically active Flag-SENP1 (WT) or inactive Flag-SENP1 (MT), and SUMOylation assay was performed to isolate SUMOylated proteins. PIM1 SUMOylation was analyzed by western blotting using PIM1 (12H8) antibody. Western blotting of whole cell lysate or input was done using Flag-tag antibody to confirm expression of SENP1. Empty vector was included, where appropriate, to maintain equal amounts of transfected plasmid DNA. ( f ) Lysates from K562 cells (treated with 20 μM MG132 for 6 hours) were incubated at 30 °C for 30 minutes in the absence or presence of 50 nM recombinant catalytic domain of SUMO protease, SENP1, followed by western blotting using PIM1 (12H8) antibody.
    Figure Legend Snippet: SUMOylation of PIM1 in vitro and in cultured cells. ( a ) In vitro transcribed and translated 35 S-methionine labeled PIM1 was incubated with recombinant SAE1/2, UBC9 with SUMO1 or SUMO2 in the presence of ATP-regeneration system. SP100 was used as a positive control in the SUMOylation reaction. SUMOylation of radiolabelled PIM1 was visualized on a Phosphorimager. ( b ) Bacterially expressed and purified GST-PIM1 was incubated in the presence of ATP, recombinant SAE1/2, UBC9 with SUMO1 (left) or SUMO2 (right). SUMOylated PIM1 was detected by western blotting using a GST-tag antibody. ( c ) COS7 cells were transfected with plasmids encoding MYC-tagged PIM1 alone or in combination with 6His-SUMO1, 2 or 3 and SUMOylation assay was carried out using Ni 2+ -NTA beads, 42–48 hours post transfection. PIM1 SUMOylation was analyzed by western blotting of Ni 2+ -NTA pull-down samples using MYC-tag (9E10) antibody. Total levels of PIM1 expressed under each transfection condition were analyzed by western blotting of Input samples using MYC-tag (9E10) antibody. ( d ) H1299 cells were transfected with a plasmid expressing 6His-PIM1, and PIM1 was affinity purified under denaturing conditions as done previously for 6His-SUMO protein. Eluted proteins were analyzed by western blotting using PIM1 (12H8) and SUMO2 antibody to detect SUMOylated PIM1. ( e ) COS7 cells were transfected with plasmids expressing PIM1 alone, or with 6His-SUMO2 in combination with catalytically active Flag-SENP1 (WT) or inactive Flag-SENP1 (MT), and SUMOylation assay was performed to isolate SUMOylated proteins. PIM1 SUMOylation was analyzed by western blotting using PIM1 (12H8) antibody. Western blotting of whole cell lysate or input was done using Flag-tag antibody to confirm expression of SENP1. Empty vector was included, where appropriate, to maintain equal amounts of transfected plasmid DNA. ( f ) Lysates from K562 cells (treated with 20 μM MG132 for 6 hours) were incubated at 30 °C for 30 minutes in the absence or presence of 50 nM recombinant catalytic domain of SUMO protease, SENP1, followed by western blotting using PIM1 (12H8) antibody.

    Techniques Used: In Vitro, Cell Culture, Labeling, Incubation, Recombinant, Positive Control, Purification, Western Blot, Transfection, Plasmid Preparation, Expressing, Affinity Purification, FLAG-tag

    9) Product Images from "Histone 3.3 Participates in a Self-Sustaining Cascade of Apoptosis That Contributes to the Progression of Chronic Obstructive Pulmonary Disease"

    Article Title: Histone 3.3 Participates in a Self-Sustaining Cascade of Apoptosis That Contributes to the Progression of Chronic Obstructive Pulmonary Disease

    Journal: American Journal of Respiratory and Critical Care Medicine

    doi: 10.1164/rccm.201302-0342OC

    Impaired proteasomal degradation of hyperacetylated H3.3 in chronic obstructive pulmonary disease. ( A ) Western blots showing in vitro evaluation of H3.3 degradation with and without hyperacetylation. Both acetylated and nonacetylated histones were purified from A549 cells treated with and without histone deacetylase inhibitors. Hyperacetylation of H3.3 was confirmed by Western blotting (Lanes 1 and 2). A 20S proteasome degradation assay was performed on these two samples, which showed that hyperacetylated H3.3 was resistant to degradation (Lanes 3 and 4). Proteasomal-specific degradation of H3.3 was determined by the addition of proteasome inhibitor MG132 (Lane 5). ( B ) Comparison of the effect of H3.3 proteasomal degradation over 16 hours with and without hyperacetylation, as quantitated by Western blotting and densitometry analysis. All of the data are expressed as the mean ± SEM for at least three independent experiments. For all of the comparisons P
    Figure Legend Snippet: Impaired proteasomal degradation of hyperacetylated H3.3 in chronic obstructive pulmonary disease. ( A ) Western blots showing in vitro evaluation of H3.3 degradation with and without hyperacetylation. Both acetylated and nonacetylated histones were purified from A549 cells treated with and without histone deacetylase inhibitors. Hyperacetylation of H3.3 was confirmed by Western blotting (Lanes 1 and 2). A 20S proteasome degradation assay was performed on these two samples, which showed that hyperacetylated H3.3 was resistant to degradation (Lanes 3 and 4). Proteasomal-specific degradation of H3.3 was determined by the addition of proteasome inhibitor MG132 (Lane 5). ( B ) Comparison of the effect of H3.3 proteasomal degradation over 16 hours with and without hyperacetylation, as quantitated by Western blotting and densitometry analysis. All of the data are expressed as the mean ± SEM for at least three independent experiments. For all of the comparisons P

    Techniques Used: Western Blot, In Vitro, Purification, Histone Deacetylase Assay, Degradation Assay

    ( A ) H3.3 cytotoxicity in the primary lung cells. Indirect immunofluorescence detecting H3.3 attached to the membrane of the primary lung cells. The cells were incubated with purified recombinant H3.3 (10 μg/ml) for 1 hour and were extensively washed with phosphate-buffered saline. ( B ) The primary lung cell damage was measured 24 hours after incubation with recombinant H3.3 (10 μg/ml) using fluorescent detection of annexin V binding and propidium iodide incorporation.
    Figure Legend Snippet: ( A ) H3.3 cytotoxicity in the primary lung cells. Indirect immunofluorescence detecting H3.3 attached to the membrane of the primary lung cells. The cells were incubated with purified recombinant H3.3 (10 μg/ml) for 1 hour and were extensively washed with phosphate-buffered saline. ( B ) The primary lung cell damage was measured 24 hours after incubation with recombinant H3.3 (10 μg/ml) using fluorescent detection of annexin V binding and propidium iodide incorporation.

    Techniques Used: Immunofluorescence, Incubation, Purification, Recombinant, Binding Assay

    ( A ) Immunohistochemical analysis of the lungs of Global Initiative for Chronic Obstructive Lung Disease (GOLD) IV and ex-smokers, showing the presence of extracellular H3.3 in the lumen of the airway inside the plug of mucus together with cell debris; these free histones are attached to the cilia of the epithelial cells ( red arrowheads ). ( B ) The Western blot quantitation of H3.3 in the bronchoalveolar lavage fluid (BALF) of GOLD II and ex-smoker subjects ( see Figure E3). ( C ) Plasma H3.3 levels in subjects with GOLD IV as compared with current and never-smokers as determined by Western blot analysis ( see Figure E3). P
    Figure Legend Snippet: ( A ) Immunohistochemical analysis of the lungs of Global Initiative for Chronic Obstructive Lung Disease (GOLD) IV and ex-smokers, showing the presence of extracellular H3.3 in the lumen of the airway inside the plug of mucus together with cell debris; these free histones are attached to the cilia of the epithelial cells ( red arrowheads ). ( B ) The Western blot quantitation of H3.3 in the bronchoalveolar lavage fluid (BALF) of GOLD II and ex-smoker subjects ( see Figure E3). ( C ) Plasma H3.3 levels in subjects with GOLD IV as compared with current and never-smokers as determined by Western blot analysis ( see Figure E3). P

    Techniques Used: Immunohistochemistry, Western Blot, Quantitation Assay

    Histone 3.3 acetylation detection by slot blotting. ( A ) Detection of the expression levels of H3.3, H3 AcK10, H3 AcK19, and H3 AcK24 in individual lung lysates (n = 6, each) from Global Initiative for Chronic Obstructive Lung Disease (GOLD) IV, current smokers, never-smokers, and ex-smokers as determined by slot blot. ( B ) The total acetylation levels were determined by quantitative densitometry; the values were normalized against the total H3.3 and are shown as the average of each group. The blue diamond represent H3 AcK24, red circles H3 AcK19, and black triangles H3 AcK10; P
    Figure Legend Snippet: Histone 3.3 acetylation detection by slot blotting. ( A ) Detection of the expression levels of H3.3, H3 AcK10, H3 AcK19, and H3 AcK24 in individual lung lysates (n = 6, each) from Global Initiative for Chronic Obstructive Lung Disease (GOLD) IV, current smokers, never-smokers, and ex-smokers as determined by slot blot. ( B ) The total acetylation levels were determined by quantitative densitometry; the values were normalized against the total H3.3 and are shown as the average of each group. The blue diamond represent H3 AcK24, red circles H3 AcK19, and black triangles H3 AcK10; P

    Techniques Used: Expressing, Dot Blot

    Ribbon diagram of histone H3.3 showing a comparison of acetylated ( blue spheres ) or ubiquitylated ( red spheres ) residues in Global Initiative for Chronic Obstructive Lung Disease IV (chronic obstructive pulmonary disease [COPD]) and ex-smokers (Control), as identified by mass spectroscopy.
    Figure Legend Snippet: Ribbon diagram of histone H3.3 showing a comparison of acetylated ( blue spheres ) or ubiquitylated ( red spheres ) residues in Global Initiative for Chronic Obstructive Lung Disease IV (chronic obstructive pulmonary disease [COPD]) and ex-smokers (Control), as identified by mass spectroscopy.

    Techniques Used: Mass Spectrometry

    Changes in human endothelial cell cytoplasmic and mitochondrial matrix [Ca 2+ ] in response to H3.3 were simultaneously measured by fluo-4 and rhod-2 imaging, respectively. ( A ) Representative images of human endothelial cells showing cytosolic ( green ) and mitochondrial ( red ) [Ca 2+ ] before, during, and after histone 3.3 exposure. ( B ) Changes in cytoplasmic ( green ) and mitochondrial matrix ( red ) [Ca 2+ ] responses in response to H3.3 (10 μg). n = 3. ( C ) Representative images showing TMRE (mitochondrial membrane potential indicator) fluorescence in human endothelial cells treated with 10, 20, or 50 μg/ml of H3.3 for 24 hours. Untreated cells served as controls. ( D ) Bar chart represents the quantitation of nuclear TMRE fluorescence (indicator of membrane potential loss). n = 3.
    Figure Legend Snippet: Changes in human endothelial cell cytoplasmic and mitochondrial matrix [Ca 2+ ] in response to H3.3 were simultaneously measured by fluo-4 and rhod-2 imaging, respectively. ( A ) Representative images of human endothelial cells showing cytosolic ( green ) and mitochondrial ( red ) [Ca 2+ ] before, during, and after histone 3.3 exposure. ( B ) Changes in cytoplasmic ( green ) and mitochondrial matrix ( red ) [Ca 2+ ] responses in response to H3.3 (10 μg). n = 3. ( C ) Representative images showing TMRE (mitochondrial membrane potential indicator) fluorescence in human endothelial cells treated with 10, 20, or 50 μg/ml of H3.3 for 24 hours. Untreated cells served as controls. ( D ) Bar chart represents the quantitation of nuclear TMRE fluorescence (indicator of membrane potential loss). n = 3.

    Techniques Used: Imaging, Fluorescence, Quantitation Assay

    Comparison of gene expression levels of H3.3, H3.1, and H2B in the individual lung tissue from subjects with chronic obstructive pulmonary disease (Global Initiative for Chronic Obstructive Lung Disease [GOLD] II and IV) and control subjects (ex-smokers), as determined by the TaqMan quantitative polymerase chain reaction assay system, to measure mRNA. Gene expression levels were not different when GOLD II was compared with control subjects. Only twofold increase in H3.3 genes expression (H3F3A and H3F3B) was observed when GOLD IV was compared with the control subjects. P
    Figure Legend Snippet: Comparison of gene expression levels of H3.3, H3.1, and H2B in the individual lung tissue from subjects with chronic obstructive pulmonary disease (Global Initiative for Chronic Obstructive Lung Disease [GOLD] II and IV) and control subjects (ex-smokers), as determined by the TaqMan quantitative polymerase chain reaction assay system, to measure mRNA. Gene expression levels were not different when GOLD II was compared with control subjects. Only twofold increase in H3.3 genes expression (H3F3A and H3F3B) was observed when GOLD IV was compared with the control subjects. P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    ( A ) Western blots showing a comparison of the ubiquitination pattern of H3.3 in the chronic obstructive pulmonary disease and ex-smokers groups. ( B ) Identification of H3.3 ubiquitination by mass spectroscopy. The tandem mass spectrometry profile of each of the two identified peptides that were differentially ubiquitinated in chronic obstructive pulmonary disease and their representative sequence are shown at the top left of each panel.
    Figure Legend Snippet: ( A ) Western blots showing a comparison of the ubiquitination pattern of H3.3 in the chronic obstructive pulmonary disease and ex-smokers groups. ( B ) Identification of H3.3 ubiquitination by mass spectroscopy. The tandem mass spectrometry profile of each of the two identified peptides that were differentially ubiquitinated in chronic obstructive pulmonary disease and their representative sequence are shown at the top left of each panel.

    Techniques Used: Western Blot, Mass Spectrometry, Sequencing

    ( A ) Western blots showing the cleavage of the apoptotic markers caspase 3 and 9, and the unfolded protein response apoptotic markers CHOP, eIF2α, and GRP78, from primary cells that were treated with H3.3. The concentration of H3.3 (10 μg/ml) that was used was similar to the concentration that was present in the epithelial lining fluid of chronic obstructive pulmonary disease lungs, based on the calculations from the amounts in the bronchoalveolar lavage fluid. ( B ) Measurement of time-dependent caspase 7 activation by Western blotting of endoplasmic reticulum (ER)-enriched and cytoplasmic fractions to determine the mechanism that is implicated for H3.3 cytotoxicity. The primary cells were incubated with H3.3 (10 μg/ml) for the time period indicated and were subsequently subjected to subcellular fractionation, to separate ER-enriched and cytoplasmic fractions.
    Figure Legend Snippet: ( A ) Western blots showing the cleavage of the apoptotic markers caspase 3 and 9, and the unfolded protein response apoptotic markers CHOP, eIF2α, and GRP78, from primary cells that were treated with H3.3. The concentration of H3.3 (10 μg/ml) that was used was similar to the concentration that was present in the epithelial lining fluid of chronic obstructive pulmonary disease lungs, based on the calculations from the amounts in the bronchoalveolar lavage fluid. ( B ) Measurement of time-dependent caspase 7 activation by Western blotting of endoplasmic reticulum (ER)-enriched and cytoplasmic fractions to determine the mechanism that is implicated for H3.3 cytotoxicity. The primary cells were incubated with H3.3 (10 μg/ml) for the time period indicated and were subsequently subjected to subcellular fractionation, to separate ER-enriched and cytoplasmic fractions.

    Techniques Used: Western Blot, Concentration Assay, Activation Assay, Incubation, Fractionation

    ( A ) Comparison of the cytotoxicity of enriched H3.3 from chronic obstructive pulmonary disease (COPD) nuclear lung lysates and recombinant H3.3. The primary lung cell damage was measured by a fluorescent detection of annexin V binding, 24 hours after incubation, with H3.3 extracted from the COPD lungs and recombinant H3.3 (10 μg/ml). ( B ) The primary cells were treated with increasing concentrations of recombinant H3.3 or ( C ) human recombinant histones H2B, H3.3, and H4 (10 μg/ml) for 24 hours. Cytotoxicity was evaluated by measuring bound fluorescent annexin V using a fluorescent reader and was normalized against the control cells without histone H3.3. ( D ) Comparison of the cell death in primary lung cells endogenously overexpressing recombinant H3.3 and the cells transfected to secrete recombinant H3.3. Cells transfected with empty plasmid were used as a control. P
    Figure Legend Snippet: ( A ) Comparison of the cytotoxicity of enriched H3.3 from chronic obstructive pulmonary disease (COPD) nuclear lung lysates and recombinant H3.3. The primary lung cell damage was measured by a fluorescent detection of annexin V binding, 24 hours after incubation, with H3.3 extracted from the COPD lungs and recombinant H3.3 (10 μg/ml). ( B ) The primary cells were treated with increasing concentrations of recombinant H3.3 or ( C ) human recombinant histones H2B, H3.3, and H4 (10 μg/ml) for 24 hours. Cytotoxicity was evaluated by measuring bound fluorescent annexin V using a fluorescent reader and was normalized against the control cells without histone H3.3. ( D ) Comparison of the cell death in primary lung cells endogenously overexpressing recombinant H3.3 and the cells transfected to secrete recombinant H3.3. Cells transfected with empty plasmid were used as a control. P

    Techniques Used: Recombinant, Binding Assay, Incubation, Transfection, Plasmid Preparation

    ( A ) Identification of H3.3 acetylation by mass spectroscopy. The tandem mass spectrometry profile of two representatives of the acetylated peptides identified in chronic obstructive pulmonary disease (COPD); their sequences are shown at the top left of each panel. ( B ) The Western blots showing the expression levels of H3 AcK24 and H3.3 in individual lung lysates (n = 5) from Global Initiative for Chronic Obstructive Lung Disease IV and ex-smokers. These results confirm the mass spectrometry data in A .
    Figure Legend Snippet: ( A ) Identification of H3.3 acetylation by mass spectroscopy. The tandem mass spectrometry profile of two representatives of the acetylated peptides identified in chronic obstructive pulmonary disease (COPD); their sequences are shown at the top left of each panel. ( B ) The Western blots showing the expression levels of H3 AcK24 and H3.3 in individual lung lysates (n = 5) from Global Initiative for Chronic Obstructive Lung Disease IV and ex-smokers. These results confirm the mass spectrometry data in A .

    Techniques Used: Mass Spectrometry, Western Blot, Expressing

    10) Product Images from "Heterochromatin protein 1 gamma and I?B kinase alpha interdependence during tumour necrosis factor gene transcription elongation in activated macrophages"

    Article Title: Heterochromatin protein 1 gamma and I?B kinase alpha interdependence during tumour necrosis factor gene transcription elongation in activated macrophages

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks509

    IKKα interacts with HP1γ and histone H3.3. ( A ) IKKα kinase assay with recombinant HP1γ. Western blot has been performed with anti-IKKα, anti-HP1γS93p and anti-HP1γ. ( B ) IKKα kinase assay with recombinant IKKα and histone H3.3 in presence or absence of recombinant HP1γ (top) or HP1α (bottom). Western blot has been performed with anti-IKKα, anti-H3S10p, anti-H3.3S31p, anti-HP1γ and anti-HP1α antibodies. ( C ) eCoIP experiments with nuclear extract from RAW264.7 cells treated with LPS for 1 h. Antibodies used for immunoprecipitation are named at the top of the figure and antibodies used for western blot named on the left. Input was 5% of material used for CoIP. ( D ) In vitro CoIP experiment performed with recombinant HP1γ–GST or IKKα–GST incubated with or without ATP and immunoprecipitated with anti-HP1γ or anti-IKKα antibodies. Western blot performed with anti-HP1γ or anti-IKKα antibodies. ( E ) In vitro CoIP experiment performed with recombinant HP1α-HIS incubated with recombinant HP1γ–GST or IKKα–GST and immunoprecipitated with anti-HP1α or anti-IKKα antibodies. Western blot performed with anti-HP1α antibody.
    Figure Legend Snippet: IKKα interacts with HP1γ and histone H3.3. ( A ) IKKα kinase assay with recombinant HP1γ. Western blot has been performed with anti-IKKα, anti-HP1γS93p and anti-HP1γ. ( B ) IKKα kinase assay with recombinant IKKα and histone H3.3 in presence or absence of recombinant HP1γ (top) or HP1α (bottom). Western blot has been performed with anti-IKKα, anti-H3S10p, anti-H3.3S31p, anti-HP1γ and anti-HP1α antibodies. ( C ) eCoIP experiments with nuclear extract from RAW264.7 cells treated with LPS for 1 h. Antibodies used for immunoprecipitation are named at the top of the figure and antibodies used for western blot named on the left. Input was 5% of material used for CoIP. ( D ) In vitro CoIP experiment performed with recombinant HP1γ–GST or IKKα–GST incubated with or without ATP and immunoprecipitated with anti-HP1γ or anti-IKKα antibodies. Western blot performed with anti-HP1γ or anti-IKKα antibodies. ( E ) In vitro CoIP experiment performed with recombinant HP1α-HIS incubated with recombinant HP1γ–GST or IKKα–GST and immunoprecipitated with anti-HP1α or anti-IKKα antibodies. Western blot performed with anti-HP1α antibody.

    Techniques Used: Kinase Assay, Recombinant, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, In Vitro, Incubation

    Transcription elongation-dependent accumulation of IKKα, HP1γ and H3.3S31 phosphorylation at the TNF locus. ( A ) Quantification of total IKKα, HP1γ, H3.3S31p, H3.3 and β-actin protein levels by western blot with total cell extract, after RAW264.7 cells incubation with LPS+DRB. RAW264.7 cells treated with LPS in presence or absence of DRB for 30 min LPS (30), 30 min LPS+DRB (30+DRB), 60 min LPS (60) or 30 min LPS following by 30 min with DRB (60+DRB). ChIP performed with ( B ) anti-IKKα and ( C ) anti-HP1γ. Horizontal axis indicates primers used for the real-time PCR. Data are normalized versus input and then versus an average of control regions. Data are representative of at least three independent experiments. Error bars represent standard deviation (SD) from three independent qPCR replicates.
    Figure Legend Snippet: Transcription elongation-dependent accumulation of IKKα, HP1γ and H3.3S31 phosphorylation at the TNF locus. ( A ) Quantification of total IKKα, HP1γ, H3.3S31p, H3.3 and β-actin protein levels by western blot with total cell extract, after RAW264.7 cells incubation with LPS+DRB. RAW264.7 cells treated with LPS in presence or absence of DRB for 30 min LPS (30), 30 min LPS+DRB (30+DRB), 60 min LPS (60) or 30 min LPS following by 30 min with DRB (60+DRB). ChIP performed with ( B ) anti-IKKα and ( C ) anti-HP1γ. Horizontal axis indicates primers used for the real-time PCR. Data are normalized versus input and then versus an average of control regions. Data are representative of at least three independent experiments. Error bars represent standard deviation (SD) from three independent qPCR replicates.

    Techniques Used: Western Blot, Incubation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Standard Deviation

    Related Articles

    Immunohistochemistry:

    Article Title: Histone 3.3 Participates in a Self-Sustaining Cascade of Apoptosis That Contributes to the Progression of Chronic Obstructive Pulmonary Disease
    Article Snippet: .. The specificity of the immunohistochemistry was assessed by preincubation of the H3 antibody with 2 μg of human recombinant H3.3 (New England Biolabs) protein, and no signal was observed. .. Notably, the H3.3 staining was also present in the mucus plugs in the airway lumen and in the cell debris attached to the cilia of airway epithelial cells in severe COPD (n = 4).

    In Vitro:

    Article Title: A functional SUMO-motif in the active site of PIM1 promotes its degradation via RNF4, and stimulates protein kinase activity
    Article Snippet: .. In vitro protein kinase assay PIM1 kinase assays were carried out using recombinant 6His-PIM1 (WT or mutant) with Histone H3.3 (M2507, NEB UK), c-MYC (MRC-PPU Reagents, University of Dundee) or BAD (SRP5164, Sigma) as substrate. ..

    Mutagenesis:

    Article Title: A functional SUMO-motif in the active site of PIM1 promotes its degradation via RNF4, and stimulates protein kinase activity
    Article Snippet: .. In vitro protein kinase assay PIM1 kinase assays were carried out using recombinant 6His-PIM1 (WT or mutant) with Histone H3.3 (M2507, NEB UK), c-MYC (MRC-PPU Reagents, University of Dundee) or BAD (SRP5164, Sigma) as substrate. ..

    Protein Kinase Assay:

    Article Title: A functional SUMO-motif in the active site of PIM1 promotes its degradation via RNF4, and stimulates protein kinase activity
    Article Snippet: .. In vitro protein kinase assay PIM1 kinase assays were carried out using recombinant 6His-PIM1 (WT or mutant) with Histone H3.3 (M2507, NEB UK), c-MYC (MRC-PPU Reagents, University of Dundee) or BAD (SRP5164, Sigma) as substrate. ..

    Incubation:

    Article Title: RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome
    Article Snippet: .. Unbound RNA aptamers were transferred to a fresh tube containing (1000 pmol) of either human histone H3 or H4 (New England Biolabs, Ipswich, MA) and were incubated at 37 °C for 10 min. .. The histone-bound RNA aptamers were isolated by capturing the histones and RNAs on a nitrocellulose filter as described above.

    Kinase Assay:

    Article Title: Acetylshikonin suppressed growth of colorectal tumour tissue and cells by inhibiting the intracellular kinase, T‐lymphokine‐activated killer cell‐originated protein kinase., et al. Acetylshikonin suppressed growth of colorectal tumour tissue and cells by inhibiting the intracellular kinase, T‐lymphokine‐activated killer cell‐originated protein kinase
    Article Snippet: .. The histone H3.3 human recombinant protein (substrate of Aurora A and Aurora B, Cat#M2507S) for kinase assays was purchased from New England Biolabs, Inc. (Ipswich, MA, USA). c‐Src kinase assay was purchased from Cyclex (Andes, NY, USA, Cat#CY‐1083). .. Rabbit anti‐phosphorylated TOPK (pTOPK) (1:200, Sigma, Cat#SAB4504053, https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1994 to detect immunohistochemical was purchased from Sigma.

    Recombinant:

    Article Title: Recombinant Thrombomodulin Protects Mice against Histone-Induced Lethal Thromboembolism
    Article Snippet: .. Washed platelets resuspended in Tyrode-HEPES buffer (pH 7.35) or platelet-rich plasma anticoagulated with either sodium citrate or hirudin were stimulated with recombinant human histone H3 or H4 (New England Biolabs, Ipswich, MA), or with collagen. ..

    Article Title: Notch- and Transducin-like Enhancer of Split (TLE)-dependent Histone Deacetylation Explain Interleukin 12 (IL-12) p70 Inhibition by Zymosan
    Article Snippet: .. Recombinant histone H3.3 was from New England Biolabs (Ipswich, MA). .. Recombinant active MSK1, anti-Ser(P)-10-histone H3 (catalog no. 04-817), anti-Ac-Lys-14-histone H3 (catalog no. 07-353), anti-histone H3 (catalog no. 05-928), and PKA inhibitor peptide were from Upstate Biotechnology (Lake Placid, NY).

    Article Title: A functional SUMO-motif in the active site of PIM1 promotes its degradation via RNF4, and stimulates protein kinase activity
    Article Snippet: .. In vitro protein kinase assay PIM1 kinase assays were carried out using recombinant 6His-PIM1 (WT or mutant) with Histone H3.3 (M2507, NEB UK), c-MYC (MRC-PPU Reagents, University of Dundee) or BAD (SRP5164, Sigma) as substrate. ..

    Article Title: Acetylshikonin suppressed growth of colorectal tumour tissue and cells by inhibiting the intracellular kinase, T‐lymphokine‐activated killer cell‐originated protein kinase., et al. Acetylshikonin suppressed growth of colorectal tumour tissue and cells by inhibiting the intracellular kinase, T‐lymphokine‐activated killer cell‐originated protein kinase
    Article Snippet: .. The histone H3.3 human recombinant protein (substrate of Aurora A and Aurora B, Cat#M2507S) for kinase assays was purchased from New England Biolabs, Inc. (Ipswich, MA, USA). c‐Src kinase assay was purchased from Cyclex (Andes, NY, USA, Cat#CY‐1083). .. Rabbit anti‐phosphorylated TOPK (pTOPK) (1:200, Sigma, Cat#SAB4504053, https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1994 to detect immunohistochemical was purchased from Sigma.

    Article Title: Histone 3.3 Participates in a Self-Sustaining Cascade of Apoptosis That Contributes to the Progression of Chronic Obstructive Pulmonary Disease
    Article Snippet: .. The specificity of the immunohistochemistry was assessed by preincubation of the H3 antibody with 2 μg of human recombinant H3.3 (New England Biolabs) protein, and no signal was observed. .. Notably, the H3.3 staining was also present in the mucus plugs in the airway lumen and in the cell debris attached to the cilia of airway epithelial cells in severe COPD (n = 4).

    Article Title: Heterochromatin protein 1 gamma and I?B kinase alpha interdependence during tumour necrosis factor gene transcription elongation in activated macrophages
    Article Snippet: .. Kinase assays Recombinant proteins (100 ng) HP1γ-GST or HP1α-HIS and 1 µg of H3.3 (NEB M2507S), were added to KB. .. ATP was added where indicated to 200 µM final concentration.

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    New England Biolabs recombinant human histones h3
    Individual human <t>histones</t> H3 and H4, but not octameric core histones, trigger TG in PRP. TG performed in recalcified PRP containing individual human histone proteins was compared with that of recombinant core histone octamers (A) or purified calf thymus histones (B). CThist, mixture of purified calf thymus histones; rOctamer, octameric core histone reconstituted with recombinant human histone proteins. Panels A and B are representative of 3 independent experiments.
    Recombinant Human Histones H3, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Individual human histones H3 and H4, but not octameric core histones, trigger TG in PRP. TG performed in recalcified PRP containing individual human histone proteins was compared with that of recombinant core histone octamers (A) or purified calf thymus histones (B). CThist, mixture of purified calf thymus histones; rOctamer, octameric core histone reconstituted with recombinant human histone proteins. Panels A and B are representative of 3 independent experiments.

    Journal: Blood

    Article Title: In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps

    doi: 10.1182/blood-2016-06-722298

    Figure Lengend Snippet: Individual human histones H3 and H4, but not octameric core histones, trigger TG in PRP. TG performed in recalcified PRP containing individual human histone proteins was compared with that of recombinant core histone octamers (A) or purified calf thymus histones (B). CThist, mixture of purified calf thymus histones; rOctamer, octameric core histone reconstituted with recombinant human histone proteins. Panels A and B are representative of 3 independent experiments.

    Article Snippet: Recombinant human histones H3 and H4 were from New England Biolabs (Ipswich, MA); TRAP6 was from R & D Systems (Minneapolis, MN).

    Techniques: Recombinant, Purification

    hnDNA and individual human histone H3 and H4 trigger coagulation in plasma. TG in recalcified normal PFP (A) and normal PRP (B) containing hnDNA. TG in recalcified FXII-deficient (FXII-Def), FXI-deficient (FXI-Def), or FVII-deficient (FVII-Def) PFP in the presence or absence of 30 µg/mL of hnDNA (C). No TG was observed in any individual deficient plasma after recalcification in the absence of DNA, represented by a single flat curve (PFP no DNA, panel C). Quantification of FXIa-AT after activation of the contact system by hnDNA in the synthetic contact system activation assay as described in “Methods” (D). TG in recalcified PRP containing recombinant human histone H3 (rH3) (E) or recombinant human histone H4 (rH4) (F). Effect of citrullination of histones H3 (G) and H4 (H) on TG in recalcified PRP. All the figures are representative of at least 3 independent experiments.

    Journal: Blood

    Article Title: In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps

    doi: 10.1182/blood-2016-06-722298

    Figure Lengend Snippet: hnDNA and individual human histone H3 and H4 trigger coagulation in plasma. TG in recalcified normal PFP (A) and normal PRP (B) containing hnDNA. TG in recalcified FXII-deficient (FXII-Def), FXI-deficient (FXI-Def), or FVII-deficient (FVII-Def) PFP in the presence or absence of 30 µg/mL of hnDNA (C). No TG was observed in any individual deficient plasma after recalcification in the absence of DNA, represented by a single flat curve (PFP no DNA, panel C). Quantification of FXIa-AT after activation of the contact system by hnDNA in the synthetic contact system activation assay as described in “Methods” (D). TG in recalcified PRP containing recombinant human histone H3 (rH3) (E) or recombinant human histone H4 (rH4) (F). Effect of citrullination of histones H3 (G) and H4 (H) on TG in recalcified PRP. All the figures are representative of at least 3 independent experiments.

    Article Snippet: Recombinant human histones H3 and H4 were from New England Biolabs (Ipswich, MA); TRAP6 was from R & D Systems (Minneapolis, MN).

    Techniques: Coagulation, Activation Assay, Recombinant

    SUMOylation increase PIM1 kinase activity in vitro . ( a ) Bacterially purified 6His-PIM1 was SUMOylated in vitro using purified GST-SUMO2. Equal amounts of SUMOylated protein (including PIM1) were captured using GST-beads and incubated without or with SENP1 catalytic domain for 1 hour at 30 °C. Kinase assays were then performed using Histone H3.3 as a substrate for at 30 °C for 0, 15, 30 and 45 min. Kinase activity of SUMO2-modified or unmodified PIM1 was measured by analyzing Histone H3.3 phosphorylation using a phospho-specific antibody. Equal levels of substrate and kinase were confirmed by western blotting using indicated antibodies. ( b ) Purified WT PIM1 was first incubated with or without SENP1 catalytic domain fragment for 1 hour at 30 °C, and immediately used in a kinase assay using Histone H3.3 as substrate for 30 min at 30 °C. PIM1 kinase activity was measured by analyzing Histone H3.3 phosphorylation using a phospho-specific antibody. Equal levels of substrate and kinase were confirmed by coomassie staining of the gel.

    Journal: Scientific Reports

    Article Title: A functional SUMO-motif in the active site of PIM1 promotes its degradation via RNF4, and stimulates protein kinase activity

    doi: 10.1038/s41598-017-03775-w

    Figure Lengend Snippet: SUMOylation increase PIM1 kinase activity in vitro . ( a ) Bacterially purified 6His-PIM1 was SUMOylated in vitro using purified GST-SUMO2. Equal amounts of SUMOylated protein (including PIM1) were captured using GST-beads and incubated without or with SENP1 catalytic domain for 1 hour at 30 °C. Kinase assays were then performed using Histone H3.3 as a substrate for at 30 °C for 0, 15, 30 and 45 min. Kinase activity of SUMO2-modified or unmodified PIM1 was measured by analyzing Histone H3.3 phosphorylation using a phospho-specific antibody. Equal levels of substrate and kinase were confirmed by western blotting using indicated antibodies. ( b ) Purified WT PIM1 was first incubated with or without SENP1 catalytic domain fragment for 1 hour at 30 °C, and immediately used in a kinase assay using Histone H3.3 as substrate for 30 min at 30 °C. PIM1 kinase activity was measured by analyzing Histone H3.3 phosphorylation using a phospho-specific antibody. Equal levels of substrate and kinase were confirmed by coomassie staining of the gel.

    Article Snippet: In vitro protein kinase assay PIM1 kinase assays were carried out using recombinant 6His-PIM1 (WT or mutant) with Histone H3.3 (M2507, NEB UK), c-MYC (MRC-PPU Reagents, University of Dundee) or BAD (SRP5164, Sigma) as substrate.

    Techniques: Activity Assay, In Vitro, Purification, Incubation, Modification, Western Blot, Kinase Assay, Staining

    PIM1 SUMOylation regulates substrate specificity in vitro and in cultured cells. ( a ) 6His-PIM1 (WT or mutant) was expressed and purified from bacterial cells, and resolved by SDS-PAGE. A western blot for the same samples was also performed using a pan-phospho tyrosine antibody to detect PIM1 autophosphorylation. ( b ) The purified 6His-PIM1 proteins were treated with lambda phosphatase (+) to remove overall phosphorylation or untreated (−). Samples were resolved by SDS-PAGE, and stained with coomassie to visualize a shift in mobility, which is indicative of dephosphorylation. ( c ) In vitro kinase assays were carried out using recombinant c-MYC or Histone H3.3 as substrates, in the absence or presence of the indicated purified 6His-PIM1 proteins. The samples were resolved by SDS-PAGE, and either stained with coomassie to detect total protein levels or transferred to a nitrocellulose membrane for western blotting using phospho-specific antibodies as a measure of PIM1 kinase activity. ( d ) U2OS-FRT cells expressing YFP alone, YFP-WT PIM1 and YFP-E171A were treated with 10 ng/ml doxycycline; U2OS-FRT expressing YFP-K169R was treated with 20 ng/ml doxycycline and U2OS-FRT expressing YFP-K67M was treated with 50 ng/ml doxycycline for 48 hours, followed by western blotting using indicated antibodies.

    Journal: Scientific Reports

    Article Title: A functional SUMO-motif in the active site of PIM1 promotes its degradation via RNF4, and stimulates protein kinase activity

    doi: 10.1038/s41598-017-03775-w

    Figure Lengend Snippet: PIM1 SUMOylation regulates substrate specificity in vitro and in cultured cells. ( a ) 6His-PIM1 (WT or mutant) was expressed and purified from bacterial cells, and resolved by SDS-PAGE. A western blot for the same samples was also performed using a pan-phospho tyrosine antibody to detect PIM1 autophosphorylation. ( b ) The purified 6His-PIM1 proteins were treated with lambda phosphatase (+) to remove overall phosphorylation or untreated (−). Samples were resolved by SDS-PAGE, and stained with coomassie to visualize a shift in mobility, which is indicative of dephosphorylation. ( c ) In vitro kinase assays were carried out using recombinant c-MYC or Histone H3.3 as substrates, in the absence or presence of the indicated purified 6His-PIM1 proteins. The samples were resolved by SDS-PAGE, and either stained with coomassie to detect total protein levels or transferred to a nitrocellulose membrane for western blotting using phospho-specific antibodies as a measure of PIM1 kinase activity. ( d ) U2OS-FRT cells expressing YFP alone, YFP-WT PIM1 and YFP-E171A were treated with 10 ng/ml doxycycline; U2OS-FRT expressing YFP-K169R was treated with 20 ng/ml doxycycline and U2OS-FRT expressing YFP-K67M was treated with 50 ng/ml doxycycline for 48 hours, followed by western blotting using indicated antibodies.

    Article Snippet: In vitro protein kinase assay PIM1 kinase assays were carried out using recombinant 6His-PIM1 (WT or mutant) with Histone H3.3 (M2507, NEB UK), c-MYC (MRC-PPU Reagents, University of Dundee) or BAD (SRP5164, Sigma) as substrate.

    Techniques: In Vitro, Cell Culture, Mutagenesis, Purification, SDS Page, Western Blot, Staining, De-Phosphorylation Assay, Recombinant, Activity Assay, Expressing

    Efficacy of anti-histone aptamers when administered after histones. a Aptamer inhibition of histone-mediated cytotoxicity of endothelial cells determined by MTS assay. EA.hy926 cells treated with 200 µg mL −1 of calf thymus histones followed by administration of either vehicle (negative control), heparin (positive control, 1:1), KU7 aptamer (1:2) or KU9 aptamer (1:2) at time points of 0, 5, 10, 15, 30, 45, 60, 90, 120 and 180 min after CTH; n = 3 biological replicates; * p

    Journal: Nature Communications

    Article Title: RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome

    doi: 10.1038/s41467-018-08030-y

    Figure Lengend Snippet: Efficacy of anti-histone aptamers when administered after histones. a Aptamer inhibition of histone-mediated cytotoxicity of endothelial cells determined by MTS assay. EA.hy926 cells treated with 200 µg mL −1 of calf thymus histones followed by administration of either vehicle (negative control), heparin (positive control, 1:1), KU7 aptamer (1:2) or KU9 aptamer (1:2) at time points of 0, 5, 10, 15, 30, 45, 60, 90, 120 and 180 min after CTH; n = 3 biological replicates; * p

    Article Snippet: Unbound RNA aptamers were transferred to a fresh tube containing (1000 pmol) of either human histone H3 or H4 (New England Biolabs, Ipswich, MA) and were incubated at 37 °C for 10 min.

    Techniques: Inhibition, MTS Assay, Negative Control, Positive Control

    Aptamers bind to human neutrophil-derived NETs and inhibit NET-induced cytotoxicity. a Confocal microscopy of human neutrophil-derived NETs. Single images are shown in gray scale. DAPI (top, left); histones (top, right); aptamer KU7 (bottom, left); merged images show: cyan, DAPI labeling of DNA, yellow, histones, and magenta, aptamer KU7–647 (bottom, right). White areas in the merged image represent close proximity of DNA, histone and aptamer. Representative images, captured with 40× oil and 2.8× zoom. Scale is equivalent to 10 µm. b Aptamer inhibition of NETs-mediated cytotoxicity of endothelial cells determined by MTS assay. EA.hy926 cells treated with 8 µg per well of NETs material (based on DNA concentration) and/or 8 µg per well (10.66 µM) of aptamer (KU7); * p

    Journal: Nature Communications

    Article Title: RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome

    doi: 10.1038/s41467-018-08030-y

    Figure Lengend Snippet: Aptamers bind to human neutrophil-derived NETs and inhibit NET-induced cytotoxicity. a Confocal microscopy of human neutrophil-derived NETs. Single images are shown in gray scale. DAPI (top, left); histones (top, right); aptamer KU7 (bottom, left); merged images show: cyan, DAPI labeling of DNA, yellow, histones, and magenta, aptamer KU7–647 (bottom, right). White areas in the merged image represent close proximity of DNA, histone and aptamer. Representative images, captured with 40× oil and 2.8× zoom. Scale is equivalent to 10 µm. b Aptamer inhibition of NETs-mediated cytotoxicity of endothelial cells determined by MTS assay. EA.hy926 cells treated with 8 µg per well of NETs material (based on DNA concentration) and/or 8 µg per well (10.66 µM) of aptamer (KU7); * p

    Article Snippet: Unbound RNA aptamers were transferred to a fresh tube containing (1000 pmol) of either human histone H3 or H4 (New England Biolabs, Ipswich, MA) and were incubated at 37 °C for 10 min.

    Techniques: Derivative Assay, Confocal Microscopy, Labeling, Inhibition, MTS Assay, Concentration Assay

    Identification of histone-specific RNA aptamers using SELEX. a Schematic of the in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX) procedure. Step 1. Double-stranded DNA (DS DNA) template library (Sel2N20) is in vitro transcribed in the presence of 2′ Fluoro pyrimidines and 2’ OH purines to generate the 2′ Fluoro-modified Round 0 RNA library (RNA). Step 2. The round 0 RNA library was incubated with human albumin and human IgG to remove RNAs that bind to human serum proteins (Negative selection). Step 3. RNA bound to serum proteins was discarded. Step 4. RNA not bound to serum proteins was incubated with human histones H3 and H4, respectively. Step 5. Histone-bound aptamers were collected and reverse-transcribed into DNA. Step 6. Round 1 DNA was then transcribed into RNA for the subsequent round of selection. A total of eight rounds of selection were performed for each histone selection (see Supplementary Table 1 ). b Binding of Round 0 (R0) and Round 8 (R8) RNA to recombinant human histone H3 (top, left panel) and H4 (top, right panel) proteins. Binding of R8 RNA to human albumin (bottom, left panel) and human serum (bottom, right panel). c Percent sequence enrichment (% Enrichment) at each round of selection (black circle). The 50% sequence enrichment point (gray circle) is indicated for each selection

    Journal: Nature Communications

    Article Title: RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome

    doi: 10.1038/s41467-018-08030-y

    Figure Lengend Snippet: Identification of histone-specific RNA aptamers using SELEX. a Schematic of the in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX) procedure. Step 1. Double-stranded DNA (DS DNA) template library (Sel2N20) is in vitro transcribed in the presence of 2′ Fluoro pyrimidines and 2’ OH purines to generate the 2′ Fluoro-modified Round 0 RNA library (RNA). Step 2. The round 0 RNA library was incubated with human albumin and human IgG to remove RNAs that bind to human serum proteins (Negative selection). Step 3. RNA bound to serum proteins was discarded. Step 4. RNA not bound to serum proteins was incubated with human histones H3 and H4, respectively. Step 5. Histone-bound aptamers were collected and reverse-transcribed into DNA. Step 6. Round 1 DNA was then transcribed into RNA for the subsequent round of selection. A total of eight rounds of selection were performed for each histone selection (see Supplementary Table 1 ). b Binding of Round 0 (R0) and Round 8 (R8) RNA to recombinant human histone H3 (top, left panel) and H4 (top, right panel) proteins. Binding of R8 RNA to human albumin (bottom, left panel) and human serum (bottom, right panel). c Percent sequence enrichment (% Enrichment) at each round of selection (black circle). The 50% sequence enrichment point (gray circle) is indicated for each selection

    Article Snippet: Unbound RNA aptamers were transferred to a fresh tube containing (1000 pmol) of either human histone H3 or H4 (New England Biolabs, Ipswich, MA) and were incubated at 37 °C for 10 min.

    Techniques: In Vitro, Modification, Incubation, Selection, Binding Assay, Recombinant, Sequencing

    Binding characterization and stability measurements of individual histone RNA aptamer sequences. a Binding kinetic rate constants ( k a and K D ) determined for aptamers KU7 (left panels) and KU9 (right panels) binding to CTH (top panels), H4 (middle panels) and BSA (bottom panels). Aptamers concentrations tested: 100 nM (blue), 50 nM (black), 25 nM (red), 12.5 nM (green), 10 nM (magenta). b Serum stability measurements for aptamers KU7 and KU9 (5 μM) in 50% human serum. T 1/2 KU7 = 150 h. T 1/2 KU9 = 48 h

    Journal: Nature Communications

    Article Title: RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome

    doi: 10.1038/s41467-018-08030-y

    Figure Lengend Snippet: Binding characterization and stability measurements of individual histone RNA aptamer sequences. a Binding kinetic rate constants ( k a and K D ) determined for aptamers KU7 (left panels) and KU9 (right panels) binding to CTH (top panels), H4 (middle panels) and BSA (bottom panels). Aptamers concentrations tested: 100 nM (blue), 50 nM (black), 25 nM (red), 12.5 nM (green), 10 nM (magenta). b Serum stability measurements for aptamers KU7 and KU9 (5 μM) in 50% human serum. T 1/2 KU7 = 150 h. T 1/2 KU9 = 48 h

    Article Snippet: Unbound RNA aptamers were transferred to a fresh tube containing (1000 pmol) of either human histone H3 or H4 (New England Biolabs, Ipswich, MA) and were incubated at 37 °C for 10 min.

    Techniques: Binding Assay

    Efficacy of histone aptamer in murine model of MODS. a Survival curves of mice injected IV with CTH in the presence or absence of aptamer treatment; n = 6 per group. Molar ratio of CTH to aptamer indicated. b Weights of the liver, lung and spleen normalized to pre-treatment body weight; * p

    Journal: Nature Communications

    Article Title: RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome

    doi: 10.1038/s41467-018-08030-y

    Figure Lengend Snippet: Efficacy of histone aptamer in murine model of MODS. a Survival curves of mice injected IV with CTH in the presence or absence of aptamer treatment; n = 6 per group. Molar ratio of CTH to aptamer indicated. b Weights of the liver, lung and spleen normalized to pre-treatment body weight; * p

    Article Snippet: Unbound RNA aptamers were transferred to a fresh tube containing (1000 pmol) of either human histone H3 or H4 (New England Biolabs, Ipswich, MA) and were incubated at 37 °C for 10 min.

    Techniques: Mouse Assay, Injection

    In vitro efficacy of RNA aptamers. a Human platelet aggregation measurements using platelets derived from three independent healthy donors. Collagen (Col), histone H4 (H4), histone aptamers (KU7 and KU9), calf thymus histones (CTH), heparin (Hep); * p

    Journal: Nature Communications

    Article Title: RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome

    doi: 10.1038/s41467-018-08030-y

    Figure Lengend Snippet: In vitro efficacy of RNA aptamers. a Human platelet aggregation measurements using platelets derived from three independent healthy donors. Collagen (Col), histone H4 (H4), histone aptamers (KU7 and KU9), calf thymus histones (CTH), heparin (Hep); * p

    Article Snippet: Unbound RNA aptamers were transferred to a fresh tube containing (1000 pmol) of either human histone H3 or H4 (New England Biolabs, Ipswich, MA) and were incubated at 37 °C for 10 min.

    Techniques: In Vitro, Derivative Assay

    Efficacy of anti-histone aptamers when administered after histones. a Aptamer inhibition of histone-mediated cytotoxicity of endothelial cells determined by MTS assay. EA.hy926 cells treated with 200 µg mL −1 of calf thymus histones followed by administration of either vehicle (negative control), heparin (positive control, 1:1), KU7 aptamer (1:2) or KU9 aptamer (1:2) at time points of 0, 5, 10, 15, 30, 45, 60, 90, 120 and 180 min after CTH; n = 3 biological replicates; * p

    Journal: Nature Communications

    Article Title: RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome

    doi: 10.1038/s41467-018-08030-y

    Figure Lengend Snippet: Efficacy of anti-histone aptamers when administered after histones. a Aptamer inhibition of histone-mediated cytotoxicity of endothelial cells determined by MTS assay. EA.hy926 cells treated with 200 µg mL −1 of calf thymus histones followed by administration of either vehicle (negative control), heparin (positive control, 1:1), KU7 aptamer (1:2) or KU9 aptamer (1:2) at time points of 0, 5, 10, 15, 30, 45, 60, 90, 120 and 180 min after CTH; n = 3 biological replicates; * p

    Article Snippet: Unbound RNA aptamers were transferred to a fresh tube containing (1000 pmol) of either human histone H3 or H4 (New England Biolabs, Ipswich, MA) and were incubated at 37 °C for 10 min.

    Techniques: Inhibition, MTS Assay, Negative Control, Positive Control

    Aptamers bind to human neutrophil-derived NETs and inhibit NET-induced cytotoxicity. a Confocal microscopy of human neutrophil-derived NETs. Single images are shown in gray scale. DAPI (top, left); histones (top, right); aptamer KU7 (bottom, left); merged images show: cyan, DAPI labeling of DNA, yellow, histones, and magenta, aptamer KU7–647 (bottom, right). White areas in the merged image represent close proximity of DNA, histone and aptamer. Representative images, captured with 40× oil and 2.8× zoom. Scale is equivalent to 10 µm. b Aptamer inhibition of NETs-mediated cytotoxicity of endothelial cells determined by MTS assay. EA.hy926 cells treated with 8 µg per well of NETs material (based on DNA concentration) and/or 8 µg per well (10.66 µM) of aptamer (KU7); * p

    Journal: Nature Communications

    Article Title: RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome

    doi: 10.1038/s41467-018-08030-y

    Figure Lengend Snippet: Aptamers bind to human neutrophil-derived NETs and inhibit NET-induced cytotoxicity. a Confocal microscopy of human neutrophil-derived NETs. Single images are shown in gray scale. DAPI (top, left); histones (top, right); aptamer KU7 (bottom, left); merged images show: cyan, DAPI labeling of DNA, yellow, histones, and magenta, aptamer KU7–647 (bottom, right). White areas in the merged image represent close proximity of DNA, histone and aptamer. Representative images, captured with 40× oil and 2.8× zoom. Scale is equivalent to 10 µm. b Aptamer inhibition of NETs-mediated cytotoxicity of endothelial cells determined by MTS assay. EA.hy926 cells treated with 8 µg per well of NETs material (based on DNA concentration) and/or 8 µg per well (10.66 µM) of aptamer (KU7); * p

    Article Snippet: Unbound RNA aptamers were transferred to a fresh tube containing (1000 pmol) of either human histone H3 or H4 (New England Biolabs, Ipswich, MA) and were incubated at 37 °C for 10 min.

    Techniques: Derivative Assay, Confocal Microscopy, Labeling, Inhibition, MTS Assay, Concentration Assay

    Identification of histone-specific RNA aptamers using SELEX. a ). b Binding of Round 0 (R0) and Round 8 (R8) RNA to recombinant human histone H3 (top, left panel) and H4 (top, right panel) proteins. Binding of R8 RNA to human albumin (bottom, left panel) and human serum (bottom, right panel). c Percent sequence enrichment (% Enrichment) at each round of selection (black circle). The 50% sequence enrichment point (gray circle) is indicated for each selection

    Journal: Nature Communications

    Article Title: RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome

    doi: 10.1038/s41467-018-08030-y

    Figure Lengend Snippet: Identification of histone-specific RNA aptamers using SELEX. a ). b Binding of Round 0 (R0) and Round 8 (R8) RNA to recombinant human histone H3 (top, left panel) and H4 (top, right panel) proteins. Binding of R8 RNA to human albumin (bottom, left panel) and human serum (bottom, right panel). c Percent sequence enrichment (% Enrichment) at each round of selection (black circle). The 50% sequence enrichment point (gray circle) is indicated for each selection

    Article Snippet: Unbound RNA aptamers were transferred to a fresh tube containing (1000 pmol) of either human histone H3 or H4 (New England Biolabs, Ipswich, MA) and were incubated at 37 °C for 10 min.

    Techniques: Binding Assay, Recombinant, Sequencing, Selection

    Binding characterization and stability measurements of individual histone RNA aptamer sequences. a Binding kinetic rate constants ( k a and K D ) determined for aptamers KU7 (left panels) and KU9 (right panels) binding to CTH (top panels), H4 (middle panels) and BSA (bottom panels). Aptamers concentrations tested: 100 nM (blue), 50 nM (black), 25 nM (red), 12.5 nM (green), 10 nM (magenta). b Serum stability measurements for aptamers KU7 and KU9 (5 μM) in 50% human serum. T 1/2 KU7 = 150 h. T 1/2 KU9 = 48 h

    Journal: Nature Communications

    Article Title: RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome

    doi: 10.1038/s41467-018-08030-y

    Figure Lengend Snippet: Binding characterization and stability measurements of individual histone RNA aptamer sequences. a Binding kinetic rate constants ( k a and K D ) determined for aptamers KU7 (left panels) and KU9 (right panels) binding to CTH (top panels), H4 (middle panels) and BSA (bottom panels). Aptamers concentrations tested: 100 nM (blue), 50 nM (black), 25 nM (red), 12.5 nM (green), 10 nM (magenta). b Serum stability measurements for aptamers KU7 and KU9 (5 μM) in 50% human serum. T 1/2 KU7 = 150 h. T 1/2 KU9 = 48 h

    Article Snippet: Unbound RNA aptamers were transferred to a fresh tube containing (1000 pmol) of either human histone H3 or H4 (New England Biolabs, Ipswich, MA) and were incubated at 37 °C for 10 min.

    Techniques: Binding Assay

    Efficacy of histone aptamer in murine model of MODS. a Survival curves of mice injected IV with CTH in the presence or absence of aptamer treatment; n = 6 per group. Molar ratio of CTH to aptamer indicated. b Weights of the liver, lung and spleen normalized to pre-treatment body weight; * p

    Journal: Nature Communications

    Article Title: RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome

    doi: 10.1038/s41467-018-08030-y

    Figure Lengend Snippet: Efficacy of histone aptamer in murine model of MODS. a Survival curves of mice injected IV with CTH in the presence or absence of aptamer treatment; n = 6 per group. Molar ratio of CTH to aptamer indicated. b Weights of the liver, lung and spleen normalized to pre-treatment body weight; * p

    Article Snippet: Unbound RNA aptamers were transferred to a fresh tube containing (1000 pmol) of either human histone H3 or H4 (New England Biolabs, Ipswich, MA) and were incubated at 37 °C for 10 min.

    Techniques: Mouse Assay, Injection

    In vitro efficacy of RNA aptamers. a Human platelet aggregation measurements using platelets derived from three independent healthy donors. Collagen (Col), histone H4 (H4), histone aptamers (KU7 and KU9), calf thymus histones (CTH), heparin (Hep); * p

    Journal: Nature Communications

    Article Title: RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome

    doi: 10.1038/s41467-018-08030-y

    Figure Lengend Snippet: In vitro efficacy of RNA aptamers. a Human platelet aggregation measurements using platelets derived from three independent healthy donors. Collagen (Col), histone H4 (H4), histone aptamers (KU7 and KU9), calf thymus histones (CTH), heparin (Hep); * p

    Article Snippet: Unbound RNA aptamers were transferred to a fresh tube containing (1000 pmol) of either human histone H3 or H4 (New England Biolabs, Ipswich, MA) and were incubated at 37 °C for 10 min.

    Techniques: In Vitro, Derivative Assay