recombinant histones h3 3  (New England Biolabs)


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    New England Biolabs recombinant histones h3 3
    Recombinant Histones H3 3, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    recombinant histones h3 3  (New England Biolabs)


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    New England Biolabs recombinant histones h3 3
    Recombinant Histones H3 3, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    recombinant histone h3 1  (New England Biolabs)


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    New England Biolabs recombinant histone h3 1
    Recombinant Histone H3 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    histone h3 3  (New England Biolabs)


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    New England Biolabs histone h3 3
    Histone H3 3, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    histone h3 3  (New England Biolabs)


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    New England Biolabs histone h3 3
    Histone H3 3, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    histone h3 3  (New England Biolabs)


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    New England Biolabs histone h3 3
    Histone H3 3, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human histone h3 3  (New England Biolabs)


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    New England Biolabs human histone h3 3
    Human Histone H3 3, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human histone h3 3  (New England Biolabs)


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    New England Biolabs human histone h3 3
    Human Histone H3 3, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human histone h3 3  (New England Biolabs)


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    New England Biolabs human histone h3 3
    Human Histone H3 3, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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.
    Histone H3 3, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    histone h3 3  (New England Biolabs)


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    Histone H3 3, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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