recombinant human histones h3  (New England Biolabs)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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