histone h3 3  (New England Biolabs)


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    Histone H3 3 Human Recombinant
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    Histone H3 3 Human Recombinant 100 ug
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    DNA Binding Proteins
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    Structured Review

    New England Biolabs histone h3 3
    Histone H3 3 Human Recombinant
    Histone H3 3 Human Recombinant 100 ug
    https://www.bioz.com/result/histone h3 3/product/New England Biolabs
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    histone h3 3 - by Bioz Stars, 2020-03
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    Images

    1) 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

    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

    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

    3) 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

    4) Product Images from "The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3"

    Article Title: The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3

    Journal: Genes & Development

    doi: 10.1101/gad.566910

    Immunopurification of e-H3.1 and e-H3.3 deposition complexes from soluble nuclear fractions. ( A ) Stable expression of e-H3.1 and e-H3.3 in HeLa cells. Cells expressing e-H3.1 or e-H3.3 and control cells (CTRL) were stained with anti-HA ( top ) and DAPI ( bottom ). ( B ) Silver staining of proteins associated with e-H3.1 (lane 1 ) and e-H3.3 NCs (lane 2 ) were purified by double immunoaffinity from soluble nuclear extracts (NCs). Polypeptides identified by mass spectrometry analysis and the positions of molecular size markers are indicated. ( C ) DAXX and ATRX proteins are specific to the e-H3.3 NC. The e-H3.1 (lane 1 ) and e-H3.3 (lane 2 ) complexes were analyzed by immunoblotting with the indicated antibodies. ( D ) HIRA is not associated with H3.3. The e-H3.1 (lane 2 ) and e-H3.3 (lane 3 ) NCs were analyzed by immunoblotting with anti-HIRA antibody. (Lane 1 ) HeLa whole-cell extract was used as a control.
    Figure Legend Snippet: Immunopurification of e-H3.1 and e-H3.3 deposition complexes from soluble nuclear fractions. ( A ) Stable expression of e-H3.1 and e-H3.3 in HeLa cells. Cells expressing e-H3.1 or e-H3.3 and control cells (CTRL) were stained with anti-HA ( top ) and DAPI ( bottom ). ( B ) Silver staining of proteins associated with e-H3.1 (lane 1 ) and e-H3.3 NCs (lane 2 ) were purified by double immunoaffinity from soluble nuclear extracts (NCs). Polypeptides identified by mass spectrometry analysis and the positions of molecular size markers are indicated. ( C ) DAXX and ATRX proteins are specific to the e-H3.3 NC. The e-H3.1 (lane 1 ) and e-H3.3 (lane 2 ) complexes were analyzed by immunoblotting with the indicated antibodies. ( D ) HIRA is not associated with H3.3. The e-H3.1 (lane 2 ) and e-H3.3 (lane 3 ) NCs were analyzed by immunoblotting with anti-HIRA antibody. (Lane 1 ) HeLa whole-cell extract was used as a control.

    Techniques Used: Immu-Puri, Expressing, Staining, Silver Staining, Purification, Mass Spectrometry

    DAXX favors deposition of H3.3 in vitro. ( A ) Purification and reconstitution of recombinant DAXX/histone complex. Histones H3.1–H4 (lane 1 ) or H3.3–H4 (lane 2 ) and full-length DAXX were expressed in bacteria and mixed at equimolar ratio (lanes 3 , 4 ). ( B ) DAXX facilitates the deposition of (H3.3–H4) 2 tetramers on DNA. Negatively supercoiled DNA corresponding to topoisomer −1 was incubated with increasing amounts of (H3.3–H4) 2 tetramers (at the indicated histone/DNA ratio, rw) either in the presence (lanes 6 – 8 ) or the absence (lanes 3 – 5 ) of equimolar (to the tetramers) amounts of GST-DAXX. The reaction products were then analyzed on native 4.5% polyacrylamide gel. (Lane 1 ) Topoisomer −1 DNA. (Lane 2 ) (H3.3–H4) 2 tetrasomes reconstituted on topoisomer −1 by salt dialysis. Positions of the open circular DNA (OC), the naked topoisomer −1 DNA, and the (H3.3–H4) 2 tetrasome are indicated. ( C ) DAXX deposits more efficiently (H3.3–H4) 2 than (H3.1–H4) 2 tetramers. Topoisomer −1 was incubated with increasing amounts (at the indicated histone/DNA ratio, rw) of (H3.1–H4) 2 (lanes 3 – 5 ) or (H3.3–H4) 2 (lanes 6 – 8 ) tetramers in the presence of equimolar (to the tetramers) amounts of GST-DAXX. The reaction products were then analyzed on native 4.5% polyacrylamide gel. (Lane 1 ) Topoisomer −1 DNA. (Lane 2 ) (H3.3–H4) 2 tetrasomes reconstituted on topoisomer −1 by salt dialysis. Positions of the open circular DNA (OC), the naked topoisomer −1 DNA, and the (H3.3–H4) 2 tetrasome are indicated.
    Figure Legend Snippet: DAXX favors deposition of H3.3 in vitro. ( A ) Purification and reconstitution of recombinant DAXX/histone complex. Histones H3.1–H4 (lane 1 ) or H3.3–H4 (lane 2 ) and full-length DAXX were expressed in bacteria and mixed at equimolar ratio (lanes 3 , 4 ). ( B ) DAXX facilitates the deposition of (H3.3–H4) 2 tetramers on DNA. Negatively supercoiled DNA corresponding to topoisomer −1 was incubated with increasing amounts of (H3.3–H4) 2 tetramers (at the indicated histone/DNA ratio, rw) either in the presence (lanes 6 – 8 ) or the absence (lanes 3 – 5 ) of equimolar (to the tetramers) amounts of GST-DAXX. The reaction products were then analyzed on native 4.5% polyacrylamide gel. (Lane 1 ) Topoisomer −1 DNA. (Lane 2 ) (H3.3–H4) 2 tetrasomes reconstituted on topoisomer −1 by salt dialysis. Positions of the open circular DNA (OC), the naked topoisomer −1 DNA, and the (H3.3–H4) 2 tetrasome are indicated. ( C ) DAXX deposits more efficiently (H3.3–H4) 2 than (H3.1–H4) 2 tetramers. Topoisomer −1 was incubated with increasing amounts (at the indicated histone/DNA ratio, rw) of (H3.1–H4) 2 (lanes 3 – 5 ) or (H3.3–H4) 2 (lanes 6 – 8 ) tetramers in the presence of equimolar (to the tetramers) amounts of GST-DAXX. The reaction products were then analyzed on native 4.5% polyacrylamide gel. (Lane 1 ) Topoisomer −1 DNA. (Lane 2 ) (H3.3–H4) 2 tetrasomes reconstituted on topoisomer −1 by salt dialysis. Positions of the open circular DNA (OC), the naked topoisomer −1 DNA, and the (H3.3–H4) 2 tetrasome are indicated.

    Techniques Used: In Vitro, Purification, Recombinant, Incubation

    DAXX preferentially associates with H3.3 in vitro and in vivo. ( A ) Primary structure of DAXX. DAXX contains several putative domains: two paired amphipathic helices (PAH1 and PAH2), a coiled-coil (CC), an acidic domain (acidic), a Ser/Pro/Thr rich domain (S/P/T-rich), and an rtt106-like domain (rtt106). ( B ) DAXX preferentially associates with H3.3 in vitro. GST-DAXX, immobilized on glutathione-agarose, was incubated with recombinant histones H3.1–H4 (lanes 3 – 5 ) or H3.3–H4 (lanes 7 – 9 ). Bead-bound complexes were washed with the indicated concentration of NaCl. Eluted proteins were fractionated on SDS-PAGE and stained with colloidal blue. The input lanes (INP) represent the amount of proteins used for the pull-down. ( C ) The central part of DAXX contains a high affinity H3.3-interacting domain. N-terminal (1–302), central (302–495), and C-terminal (495–740) regions of DAXX were produced as GST fusion proteins. The fusion proteins (lanes 2 – 7 ) and GST alone (lane 1 ), immobilized on glutathione-agarose resin, were incubated with tetramers containing epitope-tagged H3.3. ( Top ) After washing with either 0.25 or 1 M NaCl, the resin-bound tetramers were analyzed by immunoblotting using anti-HA antibody. ( Bottom ) To compare the levels of the GST fusions used for the pull-down, the blot was first stained with Ponceau red. The input lane (i) represents 40% of the amount of tetramers used for the pull-down. ( D ) purified by double immunoaffinity from either cytoplasmic extract (CC) or soluble nuclear extract (NC). The polypeptides identified by mass spectrometry analysis are indicated. ( E ) The DAXX complex, but not the HIRA complex, contains H3.3. The e-DAXX (lanes 2 , 3 ) and e-HIRA (lanes 4 , 5 ) complexes were analyzed by immunoblotting with anti-HA ( top ) and anti-H3.3 ( bottom ) antibodies. (Lane 1 ) Total histones purified from HeLa cells were used as control.
    Figure Legend Snippet: DAXX preferentially associates with H3.3 in vitro and in vivo. ( A ) Primary structure of DAXX. DAXX contains several putative domains: two paired amphipathic helices (PAH1 and PAH2), a coiled-coil (CC), an acidic domain (acidic), a Ser/Pro/Thr rich domain (S/P/T-rich), and an rtt106-like domain (rtt106). ( B ) DAXX preferentially associates with H3.3 in vitro. GST-DAXX, immobilized on glutathione-agarose, was incubated with recombinant histones H3.1–H4 (lanes 3 – 5 ) or H3.3–H4 (lanes 7 – 9 ). Bead-bound complexes were washed with the indicated concentration of NaCl. Eluted proteins were fractionated on SDS-PAGE and stained with colloidal blue. The input lanes (INP) represent the amount of proteins used for the pull-down. ( C ) The central part of DAXX contains a high affinity H3.3-interacting domain. N-terminal (1–302), central (302–495), and C-terminal (495–740) regions of DAXX were produced as GST fusion proteins. The fusion proteins (lanes 2 – 7 ) and GST alone (lane 1 ), immobilized on glutathione-agarose resin, were incubated with tetramers containing epitope-tagged H3.3. ( Top ) After washing with either 0.25 or 1 M NaCl, the resin-bound tetramers were analyzed by immunoblotting using anti-HA antibody. ( Bottom ) To compare the levels of the GST fusions used for the pull-down, the blot was first stained with Ponceau red. The input lane (i) represents 40% of the amount of tetramers used for the pull-down. ( D ) purified by double immunoaffinity from either cytoplasmic extract (CC) or soluble nuclear extract (NC). The polypeptides identified by mass spectrometry analysis are indicated. ( E ) The DAXX complex, but not the HIRA complex, contains H3.3. The e-DAXX (lanes 2 , 3 ) and e-HIRA (lanes 4 , 5 ) complexes were analyzed by immunoblotting with anti-HA ( top ) and anti-H3.3 ( bottom ) antibodies. (Lane 1 ) Total histones purified from HeLa cells were used as control.

    Techniques Used: In Vitro, In Vivo, Incubation, Recombinant, Concentration Assay, SDS Page, Staining, Produced, Purification, Mass Spectrometry

    DAXX is stably associated with the H3.3, but not with the H3.1 complex. ( A ) Silver staining of nuclear e-H3.1 complex fractionated on a glycerol gradient. The e-H3.1 NC purified by double affinity was separated on a glycerol gradient. Fractions were pooled as indicated at the top of the gel. The approximate molecular weight of the different subcomplexes was estimated using the NativeMark molecular weight marker (MWM; Invitrogen). ( B ) Silver staining of nuclear e-H3.3 complex fractionated on a glycerol gradient. Experiments were performed as described in A . ( C ) Silver staining of pooled fractions containing e-H3.1 and e-H3.3 nuclear subcomplexes (LNC and HNC) and of e-H3.1 and e-H3.3 CCs. ( D ) Immunoblotting of pooled fractions containing e-H3.1 and e-H3.3 nuclear subcomplexes (LNC and HNC) and of e-H3.1 and e-H3.3 CCs with the indicated antibodies. Input fraction (extract) is shown for the blot with anti-HIRA.
    Figure Legend Snippet: DAXX is stably associated with the H3.3, but not with the H3.1 complex. ( A ) Silver staining of nuclear e-H3.1 complex fractionated on a glycerol gradient. The e-H3.1 NC purified by double affinity was separated on a glycerol gradient. Fractions were pooled as indicated at the top of the gel. The approximate molecular weight of the different subcomplexes was estimated using the NativeMark molecular weight marker (MWM; Invitrogen). ( B ) Silver staining of nuclear e-H3.3 complex fractionated on a glycerol gradient. Experiments were performed as described in A . ( C ) Silver staining of pooled fractions containing e-H3.1 and e-H3.3 nuclear subcomplexes (LNC and HNC) and of e-H3.1 and e-H3.3 CCs. ( D ) Immunoblotting of pooled fractions containing e-H3.1 and e-H3.3 nuclear subcomplexes (LNC and HNC) and of e-H3.1 and e-H3.3 CCs with the indicated antibodies. Input fraction (extract) is shown for the blot with anti-HIRA.

    Techniques Used: Stable Transfection, Silver Staining, Purification, Molecular Weight, Marker

    DAXX-dependent deposition of H3.3 on pericentric heterochromatin. ( A ) DAXX and ATRX are present on pericentric DNA repeats in wild-type MEFs. Presence of DAXX ( left panel) and ATRX ( right panel) on pericentric DNA repeats was investigated by ChIP assays using specific antibodies. (−Ab) Control sample in which primary antibody was omitted. Results are expressed as percentage of chromatin input used for immunoprecipitation. ( B ) The level of transcripts from pericentric DNA repeats is reduced in DAXX-deficient cells. Relative mRNA level for pericentric DNA repeats in wild-type and DAXX −/− MEFs was determined by quantitative RT–PCR. Results are represented as relative expression level of pericentric DNA repeats versus GAPDH . Mean ± standard deviation of four independent experiments. ( C ) Depletion of H3.3A and H3.3B resulted in a decrease in transcription from pericentric DNA repeats. MEFs were transfected with control siRNA (siCTRL) or a mixture of H3.3A and H3.3B siRNA (siH3.3). Relative mRNA levels for pericentric DNA repeats, H3.3A , and H3.3B were determined by quantitative RT–PCR. Results were normalized to GAPDH and were set at 1 in cells transfected with control siRNA. Mean ± standard deviation of three independent experiments. ( D ) DAXX is required for deposition of H3.3 onto pericentric DNA repeats outside of S phase. DAXX −/− MEFs were deprived of serum for 48 h before being cotransfected with empty vector (CTRL) or else epitope-tagged H3.1 or H3.3 expression vector in combination with DAXX expression vector where indicated. Forty hours later, cells were reinduced for 8 h with 20% FCS in the presence of aphidicolin and were subjected to ChIP assays. Results are expressed as percentage of chromatin input immunoprecipitated. Mean ± standard deviation of three independent experiments.
    Figure Legend Snippet: DAXX-dependent deposition of H3.3 on pericentric heterochromatin. ( A ) DAXX and ATRX are present on pericentric DNA repeats in wild-type MEFs. Presence of DAXX ( left panel) and ATRX ( right panel) on pericentric DNA repeats was investigated by ChIP assays using specific antibodies. (−Ab) Control sample in which primary antibody was omitted. Results are expressed as percentage of chromatin input used for immunoprecipitation. ( B ) The level of transcripts from pericentric DNA repeats is reduced in DAXX-deficient cells. Relative mRNA level for pericentric DNA repeats in wild-type and DAXX −/− MEFs was determined by quantitative RT–PCR. Results are represented as relative expression level of pericentric DNA repeats versus GAPDH . Mean ± standard deviation of four independent experiments. ( C ) Depletion of H3.3A and H3.3B resulted in a decrease in transcription from pericentric DNA repeats. MEFs were transfected with control siRNA (siCTRL) or a mixture of H3.3A and H3.3B siRNA (siH3.3). Relative mRNA levels for pericentric DNA repeats, H3.3A , and H3.3B were determined by quantitative RT–PCR. Results were normalized to GAPDH and were set at 1 in cells transfected with control siRNA. Mean ± standard deviation of three independent experiments. ( D ) DAXX is required for deposition of H3.3 onto pericentric DNA repeats outside of S phase. DAXX −/− MEFs were deprived of serum for 48 h before being cotransfected with empty vector (CTRL) or else epitope-tagged H3.1 or H3.3 expression vector in combination with DAXX expression vector where indicated. Forty hours later, cells were reinduced for 8 h with 20% FCS in the presence of aphidicolin and were subjected to ChIP assays. Results are expressed as percentage of chromatin input immunoprecipitated. Mean ± standard deviation of three independent experiments.

    Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Quantitative RT-PCR, Expressing, Standard Deviation, Transfection, Plasmid Preparation

    Related Articles

    Clone Assay:

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    Article Snippet: Human histones H3.1, H3.3, and H4 cDNA sequences were PCR-amplified by using Vent-DNA polymerase (New England Biolabs). .. All of the histones were cloned in a homemade bicistronic pET28b vector (Clontech).

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: Paragraph title: Gene cloning, site-directed mutagenesis, protein expression, and antibody production. ... Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    Amplification:

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: The orf158L gene was PCR amplified and cloned into pET-15b (Novagen). .. Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    Filtration:

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
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    Mass Spectrometry:

    Article Title: Aurora A, MCAK, and Kif18b promote Eg5-independent spindle formation
    Article Snippet: Kinase assay using recombinant His-Aurora A and recombinant human histone-3 (NEB) served as a control. .. Phosphorylation sites on Kif15 were identified by mass-spectrometry with a nano-LC-LTQ-Orbitrap (Thermo Scientific).

    Autoradiography:

    Article Title: TIE2-mediated tyrosine phosphorylation of H4 regulates DNA damage response by recruiting ABL1
    Article Snippet: The following substrates were used in this study: core histone for calf thymus (Roche, no. 10223565001), histone H3.3 (New England Biolabs, no. M2507S), and histone H4 (New England Biolabs, no. M2504S). .. Phosphorylated histone proteins were detected by autoradiography or Western blotting.

    Blocking Assay:

    Article Title: Mapping of histone-binding sites in histone replacement-completed spermatozoa
    Article Snippet: After blocking by PBS containing 3% BSA, the membrane was incubated with H3 antibody (1:3000–10,000 dilution; ab1791, Abcam) or H4 antibody (1:1000 dilution; ab10158, Abcam) and subsequently with peroxidase-conjugated anti-rabbit antibody (1:4000 dilution; Invitrogen). .. Recombinant proteins H3.1 (M2503S, NEB) and H3.3 (M2507S, NEB) were analyzed for checking sensitivities of H3 antibody to H3.1 and H3.3.

    Incubation:

    Article Title: Aurora A, MCAK, and Kif18b promote Eg5-independent spindle formation
    Article Snippet: Identification of phosphorylation sites Five micrograms of recombinant mouse His-GFP-Kif15, purified from SF9 cells was incubated with 0.75 μg recombinant human His-Aurora A (Enzo Lifesciences) for 30 min in kinase buffer (50 mM Tris pH 7.5, 15 mM MgCl, 2 mM EGTA, 0.5 mM Vanadate, 1 mM DTT) in the presence of 60 μM ATP. .. Kinase assay using recombinant His-Aurora A and recombinant human histone-3 (NEB) served as a control.

    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. .. Reactions were incubated at 30 °C for 30 min, and terminated by addition of 2X SDS sample buffer (with DTT).

    Article Title: Mapping of histone-binding sites in histone replacement-completed spermatozoa
    Article Snippet: After blocking by PBS containing 3% BSA, the membrane was incubated with H3 antibody (1:3000–10,000 dilution; ab1791, Abcam) or H4 antibody (1:1000 dilution; ab10158, Abcam) and subsequently with peroxidase-conjugated anti-rabbit antibody (1:4000 dilution; Invitrogen). .. Recombinant proteins H3.1 (M2503S, NEB) and H3.3 (M2507S, NEB) were analyzed for checking sensitivities of H3 antibody to H3.1 and H3.3.

    Expressing:

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: Paragraph title: Gene cloning, site-directed mutagenesis, protein expression, and antibody production. ... Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    Modification:

    Article Title: NAD+-SIRT1 control of H3K4 trimethylation through circadian deacetylation of MLL1
    Article Snippet: Cells were harvested in modified RIPA buffer supplemented with deacetylase inhibitors. .. Briefly, reactions were set up in a final volume of 30 μl of KMT buffer (50 mM Tris-HCl pH 8, 5 mM MgCl2 , 4 mM DTT) in the presence of 1.2 μl of S-adenosyl-L-[methyl–3 H]-methionine (Perkin-Elmer NET155H001MC) and 1μg of recombinant histone H3.3 (New England Biolabs #M2507S).

    Western Blot:

    Article Title: TIE2-mediated tyrosine phosphorylation of H4 regulates DNA damage response by recruiting ABL1
    Article Snippet: The following substrates were used in this study: core histone for calf thymus (Roche, no. 10223565001), histone H3.3 (New England Biolabs, no. M2507S), and histone H4 (New England Biolabs, no. M2504S). .. Phosphorylated histone proteins were detected by autoradiography or Western blotting.

    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. .. Phosphorylated proteins were detected by western blotting using phospho-specific antibodies.

    Article Title: Mapping of histone-binding sites in histone replacement-completed spermatozoa
    Article Snippet: Paragraph title: Western blotting ... Recombinant proteins H3.1 (M2503S, NEB) and H3.3 (M2507S, NEB) were analyzed for checking sensitivities of H3 antibody to H3.1 and H3.3.

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

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
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    Kinase Assay:

    Article Title: Aurora A, MCAK, and Kif18b promote Eg5-independent spindle formation
    Article Snippet: .. Kinase assay using recombinant His-Aurora A and recombinant human histone-3 (NEB) served as a control. .. Phosphorylation sites on Kif15 were identified by mass-spectrometry with a nano-LC-LTQ-Orbitrap (Thermo Scientific).

    Article Title: TIE2-mediated tyrosine phosphorylation of H4 regulates DNA damage response by recruiting ABL1
    Article Snippet: Paragraph title: In vitro kinase assay ... The following substrates were used in this study: core histone for calf thymus (Roche, no. 10223565001), histone H3.3 (New England Biolabs, no. M2507S), and histone H4 (New England Biolabs, no. M2504S).

    Transfection:

    Article Title: NAD+-SIRT1 control of H3K4 trimethylation through circadian deacetylation of MLL1
    Article Snippet: After 12 hours transfection, cells were treated either with the small molecule inhibitor of SIRT1, EX527 (final concentration 50 μM) or with the vehicle (ethanol) during 24 hours. .. Briefly, reactions were set up in a final volume of 30 μl of KMT buffer (50 mM Tris-HCl pH 8, 5 mM MgCl2 , 4 mM DTT) in the presence of 1.2 μl of S-adenosyl-L-[methyl–3 H]-methionine (Perkin-Elmer NET155H001MC) and 1μg of recombinant histone H3.3 (New England Biolabs #M2507S).

    Chromatography:

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
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    Immunoprecipitation:

    Article Title: NAD+-SIRT1 control of H3K4 trimethylation through circadian deacetylation of MLL1
    Article Snippet: The immunoprecipitated Flag–MLL1–myc was used for the methyltransferase assay following the protocol described in , with some modifications. .. Briefly, reactions were set up in a final volume of 30 μl of KMT buffer (50 mM Tris-HCl pH 8, 5 mM MgCl2 , 4 mM DTT) in the presence of 1.2 μl of S-adenosyl-L-[methyl–3 H]-methionine (Perkin-Elmer NET155H001MC) and 1μg of recombinant histone H3.3 (New England Biolabs #M2507S).

    Transmission Assay:

    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. .. Platelet aggregation was optically monitored by a light transmission aggregometer (MCM Hema Tracer 313M; SSR Engineering, Tokyo, Japan).

    Polymerase Chain Reaction:

    Article Title: Dataset on the effects of spermidine on linking number differences between histone H1-free and histone H1-bound circular polynucleosomes
    Article Snippet: 2.4 Materials Recombinant human histone H2A/H2B dimer, histone H3.1/H4 tetramer, histone H10 , pBR322 vector, Nt.BspQI nicking endonuclease, T4 DNA ligase and Proteinase K were purchased from New England Biolabs Inc. (Singapore). .. QIAquick PCR Purification Kit was obtained from QIAGEN Singapore Pte.

    Article Title: The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3
    Article Snippet: .. Human histones H3.1, H3.3, and H4 cDNA sequences were PCR-amplified by using Vent-DNA polymerase (New England Biolabs). .. All of the histones were cloned in a homemade bicistronic pET28b vector (Clontech).

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: The orf158L gene was PCR amplified and cloned into pET-15b (Novagen). .. Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    Recombinant:

    Article Title: Aurora A, MCAK, and Kif18b promote Eg5-independent spindle formation
    Article Snippet: .. Kinase assay using recombinant His-Aurora A and recombinant human histone-3 (NEB) served as a control. .. Phosphorylation sites on Kif15 were identified by mass-spectrometry with a nano-LC-LTQ-Orbitrap (Thermo Scientific).

    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: 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: In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps
    Article Snippet: .. Recombinant human histones H3 and H4 were from New England Biolabs (Ipswich, MA); TRAP6 was from R & D Systems (Minneapolis, MN). .. The primary rabbit polyclonal antibody against citrullinated histone H3 was from Abcam (Cambridge, MA), whereas the DyLight 594 Goat Anti-Rabbit IgG Antibody was from Vector Laboratories (Burlingame, CA).

    Article Title: Dataset on the effects of spermidine on linking number differences between histone H1-free and histone H1-bound circular polynucleosomes
    Article Snippet: .. 2.4 Materials Recombinant human histone H2A/H2B dimer, histone H3.1/H4 tetramer, histone H10 , pBR322 vector, Nt.BspQI nicking endonuclease, T4 DNA ligase and Proteinase K were purchased from New England Biolabs Inc. (Singapore). .. QIAquick PCR Purification Kit was obtained from QIAGEN Singapore Pte.

    Article Title: Mapping of histone-binding sites in histone replacement-completed spermatozoa
    Article Snippet: .. Recombinant proteins H3.1 (M2503S, NEB) and H3.3 (M2507S, NEB) were analyzed for checking sensitivities of H3 antibody to H3.1 and H3.3. ..

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: The recombinant protein was first purified using an Ni-nitrilotriacetic acid (NTA) column (Qiagen) under native conditions and then by gel filtration chromatography using a Superdex 26/75 column (Amersham). .. Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    Article Title: NAD+-SIRT1 control of H3K4 trimethylation through circadian deacetylation of MLL1
    Article Snippet: .. Briefly, reactions were set up in a final volume of 30 μl of KMT buffer (50 mM Tris-HCl pH 8, 5 mM MgCl2 , 4 mM DTT) in the presence of 1.2 μl of S-adenosyl-L-[methyl–3 H]-methionine (Perkin-Elmer NET155H001MC) and 1μg of recombinant histone H3.3 (New England Biolabs #M2507S). .. 10 μl of each reaction were spotted onto Whatman P-81 paper circles (Whatman #3698-325) and processed for liquid scintillation counting as in .

    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.

    Fluorescence:

    Article Title: Mapping of histone-binding sites in histone replacement-completed spermatozoa
    Article Snippet: Chemical fluorescence signal was activated by ECL + (PerkinElmer), and the image was scanned with Odyssey systems (LI-COR). .. Recombinant proteins H3.1 (M2503S, NEB) and H3.3 (M2507S, NEB) were analyzed for checking sensitivities of H3 antibody to H3.1 and H3.3.

    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. ..

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: Paragraph title: Gene cloning, site-directed mutagenesis, protein expression, and antibody production. ... Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    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. ..

    Purification:

    Article Title: Aurora A, MCAK, and Kif18b promote Eg5-independent spindle formation
    Article Snippet: Identification of phosphorylation sites Five micrograms of recombinant mouse His-GFP-Kif15, purified from SF9 cells was incubated with 0.75 μg recombinant human His-Aurora A (Enzo Lifesciences) for 30 min in kinase buffer (50 mM Tris pH 7.5, 15 mM MgCl, 2 mM EGTA, 0.5 mM Vanadate, 1 mM DTT) in the presence of 60 μM ATP. .. Kinase assay using recombinant His-Aurora A and recombinant human histone-3 (NEB) served as a control.

    Article Title: TIE2-mediated tyrosine phosphorylation of H4 regulates DNA damage response by recruiting ABL1
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    Article Title: Dataset on the effects of spermidine on linking number differences between histone H1-free and histone H1-bound circular polynucleosomes
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    Article Title: Mapping of histone-binding sites in histone replacement-completed spermatozoa
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    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: The H3/H4 histone complex was purified from vector pET11a-H3H4 as described by English et al. ( ). .. Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    Coagulation:

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

    Article Title: TIE2-mediated tyrosine phosphorylation of H4 regulates DNA damage response by recruiting ABL1
    Article Snippet: The following substrates were used in this study: core histone for calf thymus (Roche, no. 10223565001), histone H3.3 (New England Biolabs, no. M2507S), and histone H4 (New England Biolabs, no. M2504S). .. The kinase reaction was stopped by the addition of 4× protein sample buffer (Invitrogen) and boiled for 5 min, followed by SDS-PAGE (16%) analyses.

    Plasmid Preparation:

    Article Title: In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps
    Article Snippet: Recombinant human histones H3 and H4 were from New England Biolabs (Ipswich, MA); TRAP6 was from R & D Systems (Minneapolis, MN). .. Recombinant human histones H3 and H4 were from New England Biolabs (Ipswich, MA); TRAP6 was from R & D Systems (Minneapolis, MN).

    Article Title: Dataset on the effects of spermidine on linking number differences between histone H1-free and histone H1-bound circular polynucleosomes
    Article Snippet: .. 2.4 Materials Recombinant human histone H2A/H2B dimer, histone H3.1/H4 tetramer, histone H10 , pBR322 vector, Nt.BspQI nicking endonuclease, T4 DNA ligase and Proteinase K were purchased from New England Biolabs Inc. (Singapore). .. QIAquick PCR Purification Kit was obtained from QIAGEN Singapore Pte.

    Article Title: The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3
    Article Snippet: Human histones H3.1, H3.3, and H4 cDNA sequences were PCR-amplified by using Vent-DNA polymerase (New England Biolabs). .. All of the histones were cloned in a homemade bicistronic pET28b vector (Clontech).

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: The H3/H4 histone complex was purified from vector pET11a-H3H4 as described by English et al. ( ). .. Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    Positron Emission Tomography:

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: The orf158L gene was PCR amplified and cloned into pET-15b (Novagen). .. Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    In Vitro:

    Article Title: TIE2-mediated tyrosine phosphorylation of H4 regulates DNA damage response by recruiting ABL1
    Article Snippet: Paragraph title: In vitro kinase assay ... The following substrates were used in this study: core histone for calf thymus (Roche, no. 10223565001), histone H3.3 (New England Biolabs, no. M2507S), and histone H4 (New England Biolabs, no. M2504S).

    Article Title: Recombinant Thrombomodulin Protects Mice against Histone-Induced Lethal Thromboembolism
    Article Snippet: Paragraph title: In vitro platelet aggregation assays ... 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: 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 Binding:

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: Purified ORF158L was used for generation of monoclonal antibodies in mouse, crystallization, and protein-binding assays. .. Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    Histone Deacetylase Assay:

    Article Title: NAD+-SIRT1 control of H3K4 trimethylation through circadian deacetylation of MLL1
    Article Snippet: Cells were harvested in modified RIPA buffer supplemented with deacetylase inhibitors. .. Briefly, reactions were set up in a final volume of 30 μl of KMT buffer (50 mM Tris-HCl pH 8, 5 mM MgCl2 , 4 mM DTT) in the presence of 1.2 μl of S-adenosyl-L-[methyl–3 H]-methionine (Perkin-Elmer NET155H001MC) and 1μg of recombinant histone H3.3 (New England Biolabs #M2507S).

    Concentration Assay:

    Article Title: NAD+-SIRT1 control of H3K4 trimethylation through circadian deacetylation of MLL1
    Article Snippet: After 12 hours transfection, cells were treated either with the small molecule inhibitor of SIRT1, EX527 (final concentration 50 μM) or with the vehicle (ethanol) during 24 hours. .. Briefly, reactions were set up in a final volume of 30 μl of KMT buffer (50 mM Tris-HCl pH 8, 5 mM MgCl2 , 4 mM DTT) in the presence of 1.2 μl of S-adenosyl-L-[methyl–3 H]-methionine (Perkin-Elmer NET155H001MC) and 1μg of recombinant histone H3.3 (New England Biolabs #M2507S).

    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.

    Staining:

    Article Title: In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps
    Article Snippet: Sytox Green Nucleic Acid Stain, DNAase I, streptavidin–horseradish peroxidase, and 5-thio-2-nitrobenzoic acid–peroxidase substrate and the S-2366 substrate were from ThermoFisher Scientific (Waltham, MA). .. Recombinant human histones H3 and H4 were from New England Biolabs (Ipswich, MA); TRAP6 was from R & D Systems (Minneapolis, MN).

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    • histone h3 3  (New England Biolabs)
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    New England Biolabs histone h3 3
    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 <t>H3.3</t> 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.
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    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

    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

    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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0075961

    Figure Lengend 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

    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.

    Techniques: 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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0075961

    Figure Lengend 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

    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.

    Techniques: 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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0075961

    Figure Lengend 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

    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.

    Techniques: 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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0075961

    Figure Lengend 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

    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.

    Techniques: 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).

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0075961

    Figure Lengend 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).

    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.

    Techniques: 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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0075961

    Figure Lengend 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.

    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.

    Techniques: Enzyme-linked Immunosorbent Assay

    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gks509

    Figure Lengend 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.

    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.

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gks509

    Figure Lengend 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.

    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.

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