recombinant histone 4  (New England Biolabs)


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    Name:
    Histone H4 Human Recombinant
    Description:
    Histone H4 Human Recombinant 100 ug
    Catalog Number:
    m2504s
    Price:
    82
    Size:
    100 ug
    Category:
    DNA Binding Proteins
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    Structured Review

    New England Biolabs recombinant histone 4
    Histone H4 Human Recombinant
    Histone H4 Human Recombinant 100 ug
    https://www.bioz.com/result/recombinant histone 4/product/New England Biolabs
    Average 95 stars, based on 52 article reviews
    Price from $9.99 to $1999.99
    recombinant histone 4 - by Bioz Stars, 2020-10
    95/100 stars

    Images

    1) Product Images from "SMYD2-Mediated Histone Methylation Contributes to HIV-1 Latency"

    Article Title: SMYD2-Mediated Histone Methylation Contributes to HIV-1 Latency

    Journal: Cell host & microbe

    doi: 10.1016/j.chom.2017.04.011

    SMYD2 methylates histone 4 at lysine 20 In vitro methylation assays, including histones isolated from HEK293T, recombinant full-length histone H4 or two short synthetic histone H4 peptides (aa 1–21 and aa 15–24) that were incubated with recombinant SMYD2 enzyme and radiolabeled H 3 -S-adenosyl-L-methionine (SAM) in the presence or absence of AZ391. Reactions were resolved by gel electrophoresis and developed by autoradiography. (a) In vitro methylation assays of histones isolated from HEK293T cells. (b) In vitro SMYD2 methylation assay of recombinant full-length histone H4, with or without AZ391. (c) In vitro SMYD2 methylation assays of synthetic histone H4 peptides (aa 1–21, left, and aa 15–24, right) in the presence or absence of AZ391. (d) In vitro SMYD2 methylation assay of synthetic histone H4 peptide (aa 1–21) with or without a K20A mutation. (e) In vitro methylation assays of human recombinant histone H4 using wildtype or catalytically inactive (Y240F) SMYD2. All in vitro methylation assays of recombinant histone H4 or H4 peptides were repeated at least three times, and representative Coomassie stain (left) and autoradiography (right) are shown. (f–h) In vitro SMYD2 methylation assay of recombinant full-length histone H4 was subjected to mass spectrometry. (f) Annotated HCD MS/MS spectrum of the histone H4 LysC peptide RHRKmeVLRDIQGITK containing K20 methylation. Blue lines indicate b ions and purple lines indicate y ions, with specific ions labeled atop each peak. (g–h) Integrated MS1 intensity for the RHRKmeVLRDIQGITK peptide (g) and an unmodified histone H4 peptide (h) across different samples. Error bars indicate standard deviation between technical replicate MS analyses.
    Figure Legend Snippet: SMYD2 methylates histone 4 at lysine 20 In vitro methylation assays, including histones isolated from HEK293T, recombinant full-length histone H4 or two short synthetic histone H4 peptides (aa 1–21 and aa 15–24) that were incubated with recombinant SMYD2 enzyme and radiolabeled H 3 -S-adenosyl-L-methionine (SAM) in the presence or absence of AZ391. Reactions were resolved by gel electrophoresis and developed by autoradiography. (a) In vitro methylation assays of histones isolated from HEK293T cells. (b) In vitro SMYD2 methylation assay of recombinant full-length histone H4, with or without AZ391. (c) In vitro SMYD2 methylation assays of synthetic histone H4 peptides (aa 1–21, left, and aa 15–24, right) in the presence or absence of AZ391. (d) In vitro SMYD2 methylation assay of synthetic histone H4 peptide (aa 1–21) with or without a K20A mutation. (e) In vitro methylation assays of human recombinant histone H4 using wildtype or catalytically inactive (Y240F) SMYD2. All in vitro methylation assays of recombinant histone H4 or H4 peptides were repeated at least three times, and representative Coomassie stain (left) and autoradiography (right) are shown. (f–h) In vitro SMYD2 methylation assay of recombinant full-length histone H4 was subjected to mass spectrometry. (f) Annotated HCD MS/MS spectrum of the histone H4 LysC peptide RHRKmeVLRDIQGITK containing K20 methylation. Blue lines indicate b ions and purple lines indicate y ions, with specific ions labeled atop each peak. (g–h) Integrated MS1 intensity for the RHRKmeVLRDIQGITK peptide (g) and an unmodified histone H4 peptide (h) across different samples. Error bars indicate standard deviation between technical replicate MS analyses.

    Techniques Used: In Vitro, Methylation, Isolation, Recombinant, Incubation, Nucleic Acid Electrophoresis, Autoradiography, Mutagenesis, Staining, Mass Spectrometry, Labeling, Standard Deviation

    2) Product Images from "Pressure-assisted sample preparation for proteomic analysis"

    Article Title: Pressure-assisted sample preparation for proteomic analysis

    Journal: Analytical biochemistry

    doi: 10.1016/j.ab.2013.03.023

    Time optimization at 15 kpsi for 30, 120, and 200 min for two peptides from the N-terminal tail portion of recombinant human histone H4.
    Figure Legend Snippet: Time optimization at 15 kpsi for 30, 120, and 200 min for two peptides from the N-terminal tail portion of recombinant human histone H4.

    Techniques Used: Recombinant

    MALDI–TOF mass spectrum of recombinant human histone H4 digested using the PCT method. The numbered peaks match those generated by conventional chymotrypsin digestion, providing 95% sequence coverage, and are listed in .
    Figure Legend Snippet: MALDI–TOF mass spectrum of recombinant human histone H4 digested using the PCT method. The numbered peaks match those generated by conventional chymotrypsin digestion, providing 95% sequence coverage, and are listed in .

    Techniques Used: Recombinant, Generated, Sequencing

    Pressure optimization at 30 min for 10, 15, and 25 kpsi for two peptides from the N-terminal tail portion of recombinant human histone H4.
    Figure Legend Snippet: Pressure optimization at 30 min for 10, 15, and 25 kpsi for two peptides from the N-terminal tail portion of recombinant human histone H4.

    Techniques Used: Recombinant

    RP–HPLC chromatogram showing fractionation of histones using UV detector set at 214 nm. The inset shows an enlarged range of the peptides containing peaks assigned to respective histones.
    Figure Legend Snippet: RP–HPLC chromatogram showing fractionation of histones using UV detector set at 214 nm. The inset shows an enlarged range of the peptides containing peaks assigned to respective histones.

    Techniques Used: High Performance Liquid Chromatography, Fractionation

    Chymotryptic digestion of native histone H4 using conventional and PCT methods
    Figure Legend Snippet: Chymotryptic digestion of native histone H4 using conventional and PCT methods

    Techniques Used:

    3) Product Images from "UFL1 promotes histone H4 ufmylation and ATM activation"

    Article Title: UFL1 promotes histone H4 ufmylation and ATM activation

    Journal: Nature Communications

    doi: 10.1038/s41467-019-09175-0

    Ufmylation of histone H4 enhances trimethylation of H3K9 at DSB. a , b Analysis of H3K9 trimethylation and Suv39H1 recruitment by Chromatin IP (ChIP) at DSB sites from MDA-MB-231 ROS8 cells with indicated treatments. The y -axis represents relative enrichment of target protein binding DNA compared to input. Target sequence of UFL1 shRNA (not inducible shRNA) in a is the same as inducible sh2. The data presented are mean ± s.e.m. for n = 3 independent experiments. ** p
    Figure Legend Snippet: Ufmylation of histone H4 enhances trimethylation of H3K9 at DSB. a , b Analysis of H3K9 trimethylation and Suv39H1 recruitment by Chromatin IP (ChIP) at DSB sites from MDA-MB-231 ROS8 cells with indicated treatments. The y -axis represents relative enrichment of target protein binding DNA compared to input. Target sequence of UFL1 shRNA (not inducible shRNA) in a is the same as inducible sh2. The data presented are mean ± s.e.m. for n = 3 independent experiments. ** p

    Techniques Used: Chromatin Immunoprecipitation, Multiple Displacement Amplification, Protein Binding, Sequencing, shRNA

    Model for ATM activation by UFL1. When DNA damage occurs, UFL1 is recruited by MRN complex and monoufmylates histone H4, thus recruiting SUV39H1 to DSBs to trimethylate H3K9 thereby forming H3K9me3. Tip60 binds to H3K9me3, acetylates ATM, and promotes ATM activation. C-Abl also phosphorylates Tip60 and enhances Tip60 acetyltransferase activity. Activated ATM phosphorylates UFL1 at Ser462, and enhances its activity thereby amplifying ATM activation signal and forming a positive feedback loop
    Figure Legend Snippet: Model for ATM activation by UFL1. When DNA damage occurs, UFL1 is recruited by MRN complex and monoufmylates histone H4, thus recruiting SUV39H1 to DSBs to trimethylate H3K9 thereby forming H3K9me3. Tip60 binds to H3K9me3, acetylates ATM, and promotes ATM activation. C-Abl also phosphorylates Tip60 and enhances Tip60 acetyltransferase activity. Activated ATM phosphorylates UFL1 at Ser462, and enhances its activity thereby amplifying ATM activation signal and forming a positive feedback loop

    Techniques Used: Activation Assay, Activity Assay

    UFL1 monoufmylates histone H4 and promotes ATM activation. a Selected proteins identified by mass spectrometry from irradiated 293T cell expressing Flag-His vector or Flag-His-UFM1. N = 1 sample in each group was analyzed. The full list of identified proteins is provided in Supplementary Data 1 . Among histone proteins, only H4 is enriched in the Flag-His-UFM1 purification (9 unique/20 total peptides) compared to the Flag-His purification (6 unique/7 total peptides), suggesting that H4 might be ufmylated. b Flag-His-ufmylated proteins were purified from 293T cells before and after IR (2 Gy) after purification with nickel beads and anti-Flag agarose. The immunoprecipitates were detected with indicated antibodies. c Flag-His-ufmylated H4 was purified from control and UFL1 knockdown cells with or without 2 Gy IR and blotted with indicated antibodies. d In vitro ufmylation assay. Purified UBA5, UFC1, UFL1, UFM1, and H4 proteins were incubated together in the presence of ATP and MgCl 2 at 30 ° C for 90 min. The reaction products were probed with indicated antibodies. e Wildtype (WT) histone H4 and 11 different single lysine (K) to arginine (R) mutants were transfected into U2OS cells. Flag and His tandem purification was performed and H4 ufmylation was analyzed. f Constructs expressing WT or K31R H4 were transfected into U2OS tet-on UFL1 shRNA expressing cells, and the cells were treated with doxycycline as indicated. Thirty minutes after 2 Gy IR, the cells were harvested and blotted with indicated antibodies. g Colony formation of U2OS cells expressing WT H4 or H4K31R following IR. The data presented are mean ± s.e.m. for n = 3 independent experiments. Statistical significance was calculated using two-way ANOVA. * p
    Figure Legend Snippet: UFL1 monoufmylates histone H4 and promotes ATM activation. a Selected proteins identified by mass spectrometry from irradiated 293T cell expressing Flag-His vector or Flag-His-UFM1. N = 1 sample in each group was analyzed. The full list of identified proteins is provided in Supplementary Data 1 . Among histone proteins, only H4 is enriched in the Flag-His-UFM1 purification (9 unique/20 total peptides) compared to the Flag-His purification (6 unique/7 total peptides), suggesting that H4 might be ufmylated. b Flag-His-ufmylated proteins were purified from 293T cells before and after IR (2 Gy) after purification with nickel beads and anti-Flag agarose. The immunoprecipitates were detected with indicated antibodies. c Flag-His-ufmylated H4 was purified from control and UFL1 knockdown cells with or without 2 Gy IR and blotted with indicated antibodies. d In vitro ufmylation assay. Purified UBA5, UFC1, UFL1, UFM1, and H4 proteins were incubated together in the presence of ATP and MgCl 2 at 30 ° C for 90 min. The reaction products were probed with indicated antibodies. e Wildtype (WT) histone H4 and 11 different single lysine (K) to arginine (R) mutants were transfected into U2OS cells. Flag and His tandem purification was performed and H4 ufmylation was analyzed. f Constructs expressing WT or K31R H4 were transfected into U2OS tet-on UFL1 shRNA expressing cells, and the cells were treated with doxycycline as indicated. Thirty minutes after 2 Gy IR, the cells were harvested and blotted with indicated antibodies. g Colony formation of U2OS cells expressing WT H4 or H4K31R following IR. The data presented are mean ± s.e.m. for n = 3 independent experiments. Statistical significance was calculated using two-way ANOVA. * p

    Techniques Used: Activation Assay, Mass Spectrometry, Irradiation, Expressing, Plasmid Preparation, Purification, In Vitro, Incubation, Transfection, Construct, shRNA

    4) Product Images from "Dual effect of histone H4 on prothrombin activation"

    Article Title: Dual effect of histone H4 on prothrombin activation

    Journal: Journal of thrombosis and haemostasis : JTH

    doi: 10.1111/jth.13400

    Effect of histone H4 on prothrombin activation by factor Xa
    Figure Legend Snippet: Effect of histone H4 on prothrombin activation by factor Xa

    Techniques Used: Activation Assay

    5) Product Images from "Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis"

    Article Title: Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx563

    Esco2 binds to histone H4 to regulate the acetylation level of H4K16. ( A ) Representative images of acetylated H4K16 in control, Esco2-KD, Esco2-rescue and Esco2-W530G oocytes. Scale bar, 5 μm. ( B ) The immunofluorescence intensity of acetylated H4K16 was recorded in control, Esco2-KD, Esco2-rescue and Esco2-W530G oocytes. Data were presented as mean percentage (mean ± SEM) of at least three independent experiments. Asterisk denotes statistical difference at a P
    Figure Legend Snippet: Esco2 binds to histone H4 to regulate the acetylation level of H4K16. ( A ) Representative images of acetylated H4K16 in control, Esco2-KD, Esco2-rescue and Esco2-W530G oocytes. Scale bar, 5 μm. ( B ) The immunofluorescence intensity of acetylated H4K16 was recorded in control, Esco2-KD, Esco2-rescue and Esco2-W530G oocytes. Data were presented as mean percentage (mean ± SEM) of at least three independent experiments. Asterisk denotes statistical difference at a P

    Techniques Used: Immunofluorescence

    Esco2 acetylates histone H4 at Lys16 in vitro . ( A ) Flag purification of Esco2. Esco2 and enzymatically mutant Esco2-W530G were expressed in HEK293 cells and then purified according to the Flag purification procedure. Purified Esco2-Flag and Esco2-W530G-Flag were detected with sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by both coomassie staining and western blotting with anti-Esco2 antibody. ( B ) Commercially obtained recombinant histone H4 was identified by both coomassie staining and western blotting with anti-H4 antibody. ( C ) In vitro acetylation assay with Esco2-Flag. Recombinant histone H4 was incubated with or without purified Esco2-Flag and Ac-CoA in the acetyltransferase assay buffer at 30°C for 1 h. The reactions were analyzed by western blotting with anti-histone H4 (acetyl K16) antibody for acetylation levels of H4K16 and anti-histone H4 antibody as a loading control. ( D ) In vitro acetylation assay with Esco2-W530G-Flag. Recombinant histone H4 was incubated with or without purified Esco2-W530G-Flag and Ac-CoA in the acetyltransferase assay buffer at 30°C for 1 h. The reactions were analyzed by western blotting with anti-histone H4 (acetyl K16) antibody for acetylation levels of H4K16 and anti-histone H4 antibody as a loading control.
    Figure Legend Snippet: Esco2 acetylates histone H4 at Lys16 in vitro . ( A ) Flag purification of Esco2. Esco2 and enzymatically mutant Esco2-W530G were expressed in HEK293 cells and then purified according to the Flag purification procedure. Purified Esco2-Flag and Esco2-W530G-Flag were detected with sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by both coomassie staining and western blotting with anti-Esco2 antibody. ( B ) Commercially obtained recombinant histone H4 was identified by both coomassie staining and western blotting with anti-H4 antibody. ( C ) In vitro acetylation assay with Esco2-Flag. Recombinant histone H4 was incubated with or without purified Esco2-Flag and Ac-CoA in the acetyltransferase assay buffer at 30°C for 1 h. The reactions were analyzed by western blotting with anti-histone H4 (acetyl K16) antibody for acetylation levels of H4K16 and anti-histone H4 antibody as a loading control. ( D ) In vitro acetylation assay with Esco2-W530G-Flag. Recombinant histone H4 was incubated with or without purified Esco2-W530G-Flag and Ac-CoA in the acetyltransferase assay buffer at 30°C for 1 h. The reactions were analyzed by western blotting with anti-histone H4 (acetyl K16) antibody for acetylation levels of H4K16 and anti-histone H4 antibody as a loading control.

    Techniques Used: In Vitro, Purification, Mutagenesis, Polyacrylamide Gel Electrophoresis, Staining, Western Blot, Recombinant, Acetylation Assay, Incubation

    6) Product Images from "Nuclear Cyclin D1/CDK4 Kinase Regulates CUL4 Expression and Triggers Neoplastic Growth via Activation of the PRMT5 Methyltransferase"

    Article Title: Nuclear Cyclin D1/CDK4 Kinase Regulates CUL4 Expression and Triggers Neoplastic Growth via Activation of the PRMT5 Methyltransferase

    Journal: Cancer cell

    doi: 10.1016/j.ccr.2010.08.012

    Knockdown of Fbx4 stabilizes nuclear cyclin D1 resulting in increased PRMT5-dependent histone methylation
    Figure Legend Snippet: Knockdown of Fbx4 stabilizes nuclear cyclin D1 resulting in increased PRMT5-dependent histone methylation

    Techniques Used: Methylation

    7) Product Images from "Tousled kinase TLK1B counteracts the effect of Asf1 in inhibition of histone H3-H4 tetramer formation"

    Article Title: Tousled kinase TLK1B counteracts the effect of Asf1 in inhibition of histone H3-H4 tetramer formation

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-2-128

    A. Crosslinking of H3–H4 . Histones H3 and H4 were incubated for the indicated minutes and then crosslinked with formaldehyde before separation on a 15% SDS/PAGE. The blot was probed separately with anti-H3 and anti-H4. B. Effect of Asf1 and TLK1B on H3–H4 dimer and tetramer formation. Reactions containing the indicated combinations of H3, H4, Asf1, and TLK1B (all in equivalent amount) were crosslinked as described in Methods and immunoblotted for H4 or H3. C. Western blots for Asf1 and TLK1B. The indicated reactions as in panel B were run in duplicate lanes and immunoblotted for Asf1, H3, or TLK1B (the gel was run for a longer time than in panel B to separate the larger proteins). For antibody controls, lane 1 contained only Asf1, and lane 7 contained only TLK1B. The positions of the cross-linked complexes identified by mobility and immunoreactivity are indicated. D. TLK1B and Asf1 bind each other stoichiometrically. Reactions contained 5 μg of GST-TLK1B and varying amounts of Asf1, as indicated. After 10 min at room temperature, the samples were adsorbed on GSH-Sepharose and analyzed for bound and unbound fractions after separation on a 10% SDS/PAGE, which was stained with Coomassie blue. E. Interaction of TLK1B and Asf1 and the effect of ATP. GST-TLK1B and Asf1 (1 μg each) were incubated for 10 min at room temperature with and without 10 or 100 μM ATP, before analysis by GSH-Sepharose pull-down. The 10% SDS/PAGE gel was stained with Coomassie blue. F. MNase digestion of pBluescript assembled into pseudo-nucleosomes. In the left panel, naked supercoiled plasmid was digested with MNase for the indicated time. In the right panel, the plasmid was first incubated with equimolar H3 and H4 in high salt and then step-dialyzed as described in [ 3 ], before MNase digestion. The DNA was re-extracted from the reactions with Geneclean and run on a 1.5% agarose/TAE gel. The resulting ~120 bp ladder (a bit shorter than the repetitive 146 bp of nucleosomal DNA) is indicative of formation of a chromatinized template. The positions of the bands of a 100-bp ladder (GenRuler, Fermentas) are indicated.
    Figure Legend Snippet: A. Crosslinking of H3–H4 . Histones H3 and H4 were incubated for the indicated minutes and then crosslinked with formaldehyde before separation on a 15% SDS/PAGE. The blot was probed separately with anti-H3 and anti-H4. B. Effect of Asf1 and TLK1B on H3–H4 dimer and tetramer formation. Reactions containing the indicated combinations of H3, H4, Asf1, and TLK1B (all in equivalent amount) were crosslinked as described in Methods and immunoblotted for H4 or H3. C. Western blots for Asf1 and TLK1B. The indicated reactions as in panel B were run in duplicate lanes and immunoblotted for Asf1, H3, or TLK1B (the gel was run for a longer time than in panel B to separate the larger proteins). For antibody controls, lane 1 contained only Asf1, and lane 7 contained only TLK1B. The positions of the cross-linked complexes identified by mobility and immunoreactivity are indicated. D. TLK1B and Asf1 bind each other stoichiometrically. Reactions contained 5 μg of GST-TLK1B and varying amounts of Asf1, as indicated. After 10 min at room temperature, the samples were adsorbed on GSH-Sepharose and analyzed for bound and unbound fractions after separation on a 10% SDS/PAGE, which was stained with Coomassie blue. E. Interaction of TLK1B and Asf1 and the effect of ATP. GST-TLK1B and Asf1 (1 μg each) were incubated for 10 min at room temperature with and without 10 or 100 μM ATP, before analysis by GSH-Sepharose pull-down. The 10% SDS/PAGE gel was stained with Coomassie blue. F. MNase digestion of pBluescript assembled into pseudo-nucleosomes. In the left panel, naked supercoiled plasmid was digested with MNase for the indicated time. In the right panel, the plasmid was first incubated with equimolar H3 and H4 in high salt and then step-dialyzed as described in [ 3 ], before MNase digestion. The DNA was re-extracted from the reactions with Geneclean and run on a 1.5% agarose/TAE gel. The resulting ~120 bp ladder (a bit shorter than the repetitive 146 bp of nucleosomal DNA) is indicative of formation of a chromatinized template. The positions of the bands of a 100-bp ladder (GenRuler, Fermentas) are indicated.

    Techniques Used: Incubation, SDS Page, Western Blot, Staining, Plasmid Preparation

    8) Product Images from "PRMT7 regulates RNA-binding capacity and protein stability in Leishmania parasites"

    Article Title: PRMT7 regulates RNA-binding capacity and protein stability in Leishmania parasites

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa211

    In vitro methylation of Alba3 and RBP16 by PRMT7. ( A ) Recombinant His-PRMT7 was tested for methyltransferase activity in vitro in the presence of radioactive methyl donor S -adenosyl methionine, human histone H4 as (1 μg) a canonical RGG-containing PRMT substrate (black arrow) and a Leishmania braziliensis protein rich in arginines but devoid of RGG motifs as a negative control ( Lbr PGF2S). ( B ) Purified His-tagged PRMT7 putative substrates Alba3 WT , RBP16 WT and RGG-deficient Alba3 ΔRGG were tested, as well as Lbr PGF2S WT . PRMT7 E202 residue in the double E loop motif was mutated to K or Q to generate catalytically-inactive mutants. ( C ) RNA-binding proteins Alba3 WT and RBP16 are arginine methylated by PRMT7 in vitro , while Alba3 ΔRGG and LbrPGF2S WT are not. ( D ) In vitro methylation of target RBPs by PRMT7 is disrupted by mutating residue E202 (PRMT7 E202K and PRMT7 E202Q ). Coomassies demonstrate relative loading.
    Figure Legend Snippet: In vitro methylation of Alba3 and RBP16 by PRMT7. ( A ) Recombinant His-PRMT7 was tested for methyltransferase activity in vitro in the presence of radioactive methyl donor S -adenosyl methionine, human histone H4 as (1 μg) a canonical RGG-containing PRMT substrate (black arrow) and a Leishmania braziliensis protein rich in arginines but devoid of RGG motifs as a negative control ( Lbr PGF2S). ( B ) Purified His-tagged PRMT7 putative substrates Alba3 WT , RBP16 WT and RGG-deficient Alba3 ΔRGG were tested, as well as Lbr PGF2S WT . PRMT7 E202 residue in the double E loop motif was mutated to K or Q to generate catalytically-inactive mutants. ( C ) RNA-binding proteins Alba3 WT and RBP16 are arginine methylated by PRMT7 in vitro , while Alba3 ΔRGG and LbrPGF2S WT are not. ( D ) In vitro methylation of target RBPs by PRMT7 is disrupted by mutating residue E202 (PRMT7 E202K and PRMT7 E202Q ). Coomassies demonstrate relative loading.

    Techniques Used: In Vitro, Methylation, Recombinant, Activity Assay, Negative Control, Purification, RNA Binding Assay

    9) Product Images from "Plasmodium falciparum Histones Induce Endothelial Proinflammatory Response and Barrier Dysfunction"

    Article Title: Plasmodium falciparum Histones Induce Endothelial Proinflammatory Response and Barrier Dysfunction

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2011.11.037

    Proposed model of the induction of endothelial proinflammatory response and barrier dysfunction by P. falciparum histones. Parasite histones from merozoites are released in the proximity of endothelial cells during schizogony of cytoadherent IRBCs, where
    Figure Legend Snippet: Proposed model of the induction of endothelial proinflammatory response and barrier dysfunction by P. falciparum histones. Parasite histones from merozoites are released in the proximity of endothelial cells during schizogony of cytoadherent IRBCs, where

    Techniques Used:

    P. falciparum histone extracts induced IL-8 production through TLR2, Src family kinases, and p38 MAPK. A: Partial inhibition of IL-8 production in response to HeH by anti-TLR2, but not anti-TLR4. HDMECs in 96-well plates were preincubated with antibodies
    Figure Legend Snippet: P. falciparum histone extracts induced IL-8 production through TLR2, Src family kinases, and p38 MAPK. A: Partial inhibition of IL-8 production in response to HeH by anti-TLR2, but not anti-TLR4. HDMECs in 96-well plates were preincubated with antibodies

    Techniques Used: Inhibition

    P. falciparum histone extracts induced IL-8 production and endothelial permeability. A: HeH proteins (25 μg/lane) from merozoites of parasite clone 3D7 were separated on a 6% SDS-PAGE gel and visualized with Coomassie Blue staining. Prominent
    Figure Legend Snippet: P. falciparum histone extracts induced IL-8 production and endothelial permeability. A: HeH proteins (25 μg/lane) from merozoites of parasite clone 3D7 were separated on a 6% SDS-PAGE gel and visualized with Coomassie Blue staining. Prominent

    Techniques Used: Permeability, SDS Page, Staining

    10) Product Images from "Novel Inhibitors for PRMT1 Discovered by High-Throughput Screening Using Activity-Based Fluorescence Polarization"

    Article Title: Novel Inhibitors for PRMT1 Discovered by High-Throughput Screening Using Activity-Based Fluorescence Polarization

    Journal: ACS Chemical Biology

    doi: 10.1021/cb300024c

    5380390 and 2818500 do not inhibit SAM–dependent methyltransferases absent the SAM–binding cysteine. a) In vitro methylation of CARM1 on Histone3.1 after incubation with 10 and 100μM of potential inhibitors. b) In vitro methylation
    Figure Legend Snippet: 5380390 and 2818500 do not inhibit SAM–dependent methyltransferases absent the SAM–binding cysteine. a) In vitro methylation of CARM1 on Histone3.1 after incubation with 10 and 100μM of potential inhibitors. b) In vitro methylation

    Techniques Used: Binding Assay, In Vitro, Methylation, Incubation

    11) Product Images from "Histone levels are regulated by phosphorylation and ubiquitylation dependent proteolysis"

    Article Title: Histone levels are regulated by phosphorylation and ubiquitylation dependent proteolysis

    Journal: Nature cell biology

    doi: 10.1038/ncb1903

    Yeast cells lacking the factors involved in histone degradation accumulate excess endogenous histones bound to histone chaperones Asf1 and Cac1 (A) Endogenous histone H3 accumulates on Asf1 in a rad53 “kinase dead” mutant 38 . Asynchronous cultures of the indicated strains carrying a FLAG3 epitope on the endogenous ASF1 gene were either left untreated, or treated with the DNA replication inhibitor hydroxyurea (HU) for 90 minutes. Cells were then harvested, WCEs prepared and Asf1-FLAG3 was immunoprecipitated (IPed) as described in Methods. The indicated IPed and co-immunoprecipitated (Co-IPed) proteins were detected by Western blotting. Note that although equal amounts of Asf1-FLAG3 were IPed in all the samples, the amount of co-IPed mutant rad53K227A-TAP protein was lower compared to the wild type Rad53-TAP due to the lower stability of the mutant protein. (B) Deletion of the ubc4 , ubc5 and tom1 genes results in an accumulation of excess endogenous histone H3 bound to Asf1. Asynchronous cultures of strains expressing the histone chaperone Asf1-FLAG3 were either left untreated or treated with the DNA alkylating agent methyl methane sulfonate (MMS, 0.033%) for 90 minutes and processed exactly as described above in (A). Total histone H3 levels in the WCEs are shown to demonstrate that roughly equal amounts of extracts were used for each immunoprecipitation reaction. (C) Excess histone H3 accumulates on histone chaperone Cac1 upon the deletion of the ubc4 , ubc5 and tom1 genes. The indicated strains carrying a FLAG3 epitope on the endogenous gene encoding Cac1 were harvested and processed as described above in (A) except that no treatment with genotoxic agents such as HU or MMS were carried out. (D) Disruption of the proteasome function results in an accumulation of excess endogenous histone H3 bound to Cac1. Asynchronous cultures of the indicated temperature sensitive ( ts ) proteasome mutant strains expressing the histone chaperone Cac1-FLAG3 were grown overnight at 25°C and then shifted to the restrictive temperature of 37°C for 2.5 hours prior to harvesting the cells and processing them as described above in (C).
    Figure Legend Snippet: Yeast cells lacking the factors involved in histone degradation accumulate excess endogenous histones bound to histone chaperones Asf1 and Cac1 (A) Endogenous histone H3 accumulates on Asf1 in a rad53 “kinase dead” mutant 38 . Asynchronous cultures of the indicated strains carrying a FLAG3 epitope on the endogenous ASF1 gene were either left untreated, or treated with the DNA replication inhibitor hydroxyurea (HU) for 90 minutes. Cells were then harvested, WCEs prepared and Asf1-FLAG3 was immunoprecipitated (IPed) as described in Methods. The indicated IPed and co-immunoprecipitated (Co-IPed) proteins were detected by Western blotting. Note that although equal amounts of Asf1-FLAG3 were IPed in all the samples, the amount of co-IPed mutant rad53K227A-TAP protein was lower compared to the wild type Rad53-TAP due to the lower stability of the mutant protein. (B) Deletion of the ubc4 , ubc5 and tom1 genes results in an accumulation of excess endogenous histone H3 bound to Asf1. Asynchronous cultures of strains expressing the histone chaperone Asf1-FLAG3 were either left untreated or treated with the DNA alkylating agent methyl methane sulfonate (MMS, 0.033%) for 90 minutes and processed exactly as described above in (A). Total histone H3 levels in the WCEs are shown to demonstrate that roughly equal amounts of extracts were used for each immunoprecipitation reaction. (C) Excess histone H3 accumulates on histone chaperone Cac1 upon the deletion of the ubc4 , ubc5 and tom1 genes. The indicated strains carrying a FLAG3 epitope on the endogenous gene encoding Cac1 were harvested and processed as described above in (A) except that no treatment with genotoxic agents such as HU or MMS were carried out. (D) Disruption of the proteasome function results in an accumulation of excess endogenous histone H3 bound to Cac1. Asynchronous cultures of the indicated temperature sensitive ( ts ) proteasome mutant strains expressing the histone chaperone Cac1-FLAG3 were grown overnight at 25°C and then shifted to the restrictive temperature of 37°C for 2.5 hours prior to harvesting the cells and processing them as described above in (C).

    Techniques Used: Mutagenesis, Immunoprecipitation, Western Blot, Expressing

    Histones associated with Rad53 are ubiquitylated and the tyrosine 99 residue of histone H3 is critical for its ubiquitylation (A) Histone H3 associated with Rad53 is extensively modified. Rad53-TAP complexes were immunoprecipitated as described in Methods and analyzed by Western blotting with H3-C antibody. The migration of bulk histone H3 in WCE is indicated by the arrow. In Rad53 immunoprecipitates, multiple bands with slower mobility than the H3 band in WCE are detected, suggesting that histone H3 associated with Rad53 is heavily modified. (B) Histone H3 associated with Rad53 appears to be ubiquitylated. The Western blot shown above in (A) was stripped and reprobed with an ubiquitin antibody. The migration of free ubiquitin (Ub) is indicated by the arrow. (C) Characterization of a strain in which the only source of ubiquitin is His6-MYC tagged (MYC-Ub). WCEs prepared from wild type (WT) and MYC-Ub strains were resolved by SDS-PAGE and processed for Western blotting first with a ubiquitin (Ub) antibody (Ub Western) and, after stripping, with a MYC antibody (MYC Western). (D) Histone H4 bound to Rad53 is either multi- or poly-ubiquitylated. Histone H4 present in Rad53-TAP immunoprecipitates was analyzed by acetic acid-urea (AU) gel electrophoresis followed by Western blotting with H4 antibodies. The mobility of unmodified histone H4 is indicated by “H4” while the putative ubiquitylated histone H4 species carrying increasing numbers of ubiquitin moieties are indicated by asterisks. The mobility of H4-MYC-Ub bands is slightly retarded in the lane containing Rad53-TAP immunoprecipitates from the MYC-Ub strain compared to those derived from the WT strain. (E) Histone H3 bound to Rad53 is also either multi- or poly-ubiquitylated. Indicated samples were processed as described in (D) except that Western blotting was carried out to detect histone H3. Note that in this experiment all histone H3 appears to be modified. (F) Tyrosine 99 residue of histone H3 is required for the efficient ubiquitylation of this histone. Strains carrying FLAG-tagged endogenous genes corresponding to either the wild type (WT) or Y99F mutant histone H3 were processed as described in (D) except that Western blotting was carried out using FLAG antibodies. Note the absence of high molecular mass FLAG-H3 species in the Y99F mutant.
    Figure Legend Snippet: Histones associated with Rad53 are ubiquitylated and the tyrosine 99 residue of histone H3 is critical for its ubiquitylation (A) Histone H3 associated with Rad53 is extensively modified. Rad53-TAP complexes were immunoprecipitated as described in Methods and analyzed by Western blotting with H3-C antibody. The migration of bulk histone H3 in WCE is indicated by the arrow. In Rad53 immunoprecipitates, multiple bands with slower mobility than the H3 band in WCE are detected, suggesting that histone H3 associated with Rad53 is heavily modified. (B) Histone H3 associated with Rad53 appears to be ubiquitylated. The Western blot shown above in (A) was stripped and reprobed with an ubiquitin antibody. The migration of free ubiquitin (Ub) is indicated by the arrow. (C) Characterization of a strain in which the only source of ubiquitin is His6-MYC tagged (MYC-Ub). WCEs prepared from wild type (WT) and MYC-Ub strains were resolved by SDS-PAGE and processed for Western blotting first with a ubiquitin (Ub) antibody (Ub Western) and, after stripping, with a MYC antibody (MYC Western). (D) Histone H4 bound to Rad53 is either multi- or poly-ubiquitylated. Histone H4 present in Rad53-TAP immunoprecipitates was analyzed by acetic acid-urea (AU) gel electrophoresis followed by Western blotting with H4 antibodies. The mobility of unmodified histone H4 is indicated by “H4” while the putative ubiquitylated histone H4 species carrying increasing numbers of ubiquitin moieties are indicated by asterisks. The mobility of H4-MYC-Ub bands is slightly retarded in the lane containing Rad53-TAP immunoprecipitates from the MYC-Ub strain compared to those derived from the WT strain. (E) Histone H3 bound to Rad53 is also either multi- or poly-ubiquitylated. Indicated samples were processed as described in (D) except that Western blotting was carried out to detect histone H3. Note that in this experiment all histone H3 appears to be modified. (F) Tyrosine 99 residue of histone H3 is required for the efficient ubiquitylation of this histone. Strains carrying FLAG-tagged endogenous genes corresponding to either the wild type (WT) or Y99F mutant histone H3 were processed as described in (D) except that Western blotting was carried out using FLAG antibodies. Note the absence of high molecular mass FLAG-H3 species in the Y99F mutant.

    Techniques Used: Modification, Immunoprecipitation, Western Blot, Migration, SDS Page, Stripping Membranes, Nucleic Acid Electrophoresis, Derivative Assay, Mutagenesis

    Functional proteasomes are required for the degradation of excess histones Wild type and temperature sensitive ( ts ) proteasomal mutants carrying the pYES2-HTH- HHT2 plasmid 9 encoding galactose inducible, HIS10-TEV-HA (HTH) tagged histone H3 were used to carry out histone degradation assay essentially as described in Methods, with minor changes. The assay was carried out at the semi-permissive temperature of 30°C for the ts strains and cells were harvested every 25 minutes after switching to glucose. The levels of HTH-tagged H3 and chromosomal histone H3 were quantitated by Western blotting using the H3-C antibody 9 .
    Figure Legend Snippet: Functional proteasomes are required for the degradation of excess histones Wild type and temperature sensitive ( ts ) proteasomal mutants carrying the pYES2-HTH- HHT2 plasmid 9 encoding galactose inducible, HIS10-TEV-HA (HTH) tagged histone H3 were used to carry out histone degradation assay essentially as described in Methods, with minor changes. The assay was carried out at the semi-permissive temperature of 30°C for the ts strains and cells were harvested every 25 minutes after switching to glucose. The levels of HTH-tagged H3 and chromosomal histone H3 were quantitated by Western blotting using the H3-C antibody 9 .

    Techniques Used: Functional Assay, Plasmid Preparation, Degradation Assay, Western Blot

    Identification of the putative E2 and E3 enzymes involved in the degradation related ubiquitylation of histones (A) Yeast strains lacking Ubc4, Ubc5 and Tom1 are sensitive to histone overexpression. Wild type W303 and congenic strains carrying deletions of genes encoding E2 or E3 enzymes were transformed with either a plasmid encoding galactose inducible, HIS10-TEV-HA (HTH) tagged histone H3 (pYES2-HTH- HHT2 ) or the empty vector (pYES2-HTH) 9 . The resulting transformants were grown overnight in minimal medium lacking uracil (to select for the plasmids) and containing 2% raffinose as carbon source. 10-fold serial dilutions of each strain were plated on minimal media minus uracil, with either glucose or galactose as carbon source. The plates were incubated for 3 days at 30°C, except for the slow growing ubc4 ubc5 double mutants which were incubated for 6 days. The rad53 deletion strain carries the sml1-1 null allele to suppress the lethality due to the loss of the essential function of Rad53. (B) Disruption of the UBC4 , UBC5 and TOM1 genes causes defects in the degradation of overexpressed histones. In vivo histone degradation assay with wild type (W303) and congenic mutant strains carrying the pYES2-HTH- HHT2 plasmid were performed as described in Methods. Cells were harvested every 30 minutes after switching to glucose. The levels of HTH-tagged H3 and chromosomal histone H3 were quantitated by Western blotting using the H3-C antibody 9 .
    Figure Legend Snippet: Identification of the putative E2 and E3 enzymes involved in the degradation related ubiquitylation of histones (A) Yeast strains lacking Ubc4, Ubc5 and Tom1 are sensitive to histone overexpression. Wild type W303 and congenic strains carrying deletions of genes encoding E2 or E3 enzymes were transformed with either a plasmid encoding galactose inducible, HIS10-TEV-HA (HTH) tagged histone H3 (pYES2-HTH- HHT2 ) or the empty vector (pYES2-HTH) 9 . The resulting transformants were grown overnight in minimal medium lacking uracil (to select for the plasmids) and containing 2% raffinose as carbon source. 10-fold serial dilutions of each strain were plated on minimal media minus uracil, with either glucose or galactose as carbon source. The plates were incubated for 3 days at 30°C, except for the slow growing ubc4 ubc5 double mutants which were incubated for 6 days. The rad53 deletion strain carries the sml1-1 null allele to suppress the lethality due to the loss of the essential function of Rad53. (B) Disruption of the UBC4 , UBC5 and TOM1 genes causes defects in the degradation of overexpressed histones. In vivo histone degradation assay with wild type (W303) and congenic mutant strains carrying the pYES2-HTH- HHT2 plasmid were performed as described in Methods. Cells were harvested every 30 minutes after switching to glucose. The levels of HTH-tagged H3 and chromosomal histone H3 were quantitated by Western blotting using the H3-C antibody 9 .

    Techniques Used: Over Expression, Transformation Assay, Plasmid Preparation, Incubation, In Vivo, Degradation Assay, Mutagenesis, Western Blot

    Ubc4, Ubc5, Tom1 and Rad53 interact with each other in vivo and can ubiquitylate histones in vitro (A) The E2 enzymes Ubc4 and Ubc5 interact with Rad53 and histone H3. Asynchronous cultures of wild type (WT) strains expressing Rad53-FLAG3 and either Ubc4-MYC13 or Ubc5-MYC13 was harvested and WCEs prepared as described in Methods. Each WCE was immunoprecipitated (IPed) with MYC antibodies. The IPed material was resolved on a SDS 4–12% polyacrylamide gradient gel and processed for Western blotting to detect the IPed and co-IPed proteins. No protein was detected in the immunoprecipitated material from a WT strain lacking epitope tags (No tag). Total histone H3 levels in the WCEs are shown to demonstrate that roughly equal amounts of extracts were used for each immunoprecipitation reaction. (B) Tom1 interacts with Rad53. Asynchronous cultures of the WT strain expressing either Rad53-FLAG3 alone or in combination with Tom1-MYC13 were harvested and processed as described above for (A). Rad53-FLAG3 was IPed with FLAG antibodies and processed for Western blotting to detect the IPed and co-IPed proteins. Total Rad53-FLAG3 and histone H3 levels in the WCEs are shown to demonstrate that roughly equal amounts of extracts were used for each immunoprecipitation reaction. (C) In vitro ubiquitylation of histone H4 by E2 and E3 enzymes in a Rad53 dependent manner. Purified components were used to reconstitute the ubiquitylation of recombinant histone H4 in a Rad53 dependent manner in vitro as described in the Methods section and analyzed by Western blotting with H4 antibodies. Addition of E1 and E2 to histone H4 with or without Rad53 did not result in appreciable histone modifications (lanes 6 and 5 respectively) and neither did the addition of E3 in the absence of Rad53 (lane 7). Only the addition of Rad53 to a mixture of histone H4, ubiquitin, E1, E2 and E3 resulted in a dramatic stimulation of histone modifications, which are likely to be a mixture of ubiquitylation and phosphorylation (lane 8).
    Figure Legend Snippet: Ubc4, Ubc5, Tom1 and Rad53 interact with each other in vivo and can ubiquitylate histones in vitro (A) The E2 enzymes Ubc4 and Ubc5 interact with Rad53 and histone H3. Asynchronous cultures of wild type (WT) strains expressing Rad53-FLAG3 and either Ubc4-MYC13 or Ubc5-MYC13 was harvested and WCEs prepared as described in Methods. Each WCE was immunoprecipitated (IPed) with MYC antibodies. The IPed material was resolved on a SDS 4–12% polyacrylamide gradient gel and processed for Western blotting to detect the IPed and co-IPed proteins. No protein was detected in the immunoprecipitated material from a WT strain lacking epitope tags (No tag). Total histone H3 levels in the WCEs are shown to demonstrate that roughly equal amounts of extracts were used for each immunoprecipitation reaction. (B) Tom1 interacts with Rad53. Asynchronous cultures of the WT strain expressing either Rad53-FLAG3 alone or in combination with Tom1-MYC13 were harvested and processed as described above for (A). Rad53-FLAG3 was IPed with FLAG antibodies and processed for Western blotting to detect the IPed and co-IPed proteins. Total Rad53-FLAG3 and histone H3 levels in the WCEs are shown to demonstrate that roughly equal amounts of extracts were used for each immunoprecipitation reaction. (C) In vitro ubiquitylation of histone H4 by E2 and E3 enzymes in a Rad53 dependent manner. Purified components were used to reconstitute the ubiquitylation of recombinant histone H4 in a Rad53 dependent manner in vitro as described in the Methods section and analyzed by Western blotting with H4 antibodies. Addition of E1 and E2 to histone H4 with or without Rad53 did not result in appreciable histone modifications (lanes 6 and 5 respectively) and neither did the addition of E3 in the absence of Rad53 (lane 7). Only the addition of Rad53 to a mixture of histone H4, ubiquitin, E1, E2 and E3 resulted in a dramatic stimulation of histone modifications, which are likely to be a mixture of ubiquitylation and phosphorylation (lane 8).

    Techniques Used: In Vivo, In Vitro, Expressing, Immunoprecipitation, Western Blot, Purification, Recombinant

    Histones associated with Rad53 are phosphorylated and this modification is required for efficient histone degradation (A) Histone H3 associated with Rad53 is phosphorylated. TAP-tagged Rad53 was immunoprecipitated using IgG-Sepharose beads as described in the Methods section. Rad53-TAP bound to beads was first equilibrated with lysis buffer lacking phosphatase inhibitors and then incubated with or without 1mM ATP for 1hour at 30°C, following which the ATP treated sample was divided into three equal aliquots. One aliquot was left untreated; the second was incubated with λ-phosphatase (λ-PPase), while the third was treated with YOP tyrosine specific phosphatase (Y-PPase) for an additional hour at 30°C. All samples were boiled in SDS-PAGE sample loading buffer and proteins were resolved through a SDS 4–12% polyacrylamide gradient gel, which was processed for Western blotting with the H3-C antibody 9 . The migration of histone H3 present in whole cell extract (WCE) is indicated by the arrow. The lane labeled “No Tag” shows a mock immunoprecipitation with IgG-Sepharose performed on a strain where Rad53 was untagged. (B) Mutation of the tyrosine 99 phosphorylation site in histone H3 abolishes its degradation. Site-directed mutagenesis was used to mutate both the potential tyrosine (Y) phosphorylation sites in the H3 protein at positions 41 and 99 to phenylalanines (F), either individually (Y41F and Y99F), or in combination (Y41,99F). Histone degradation assays were carried out as described in the Methods section in wild type W303 cells bearing HTH-tagged wild type (WT) or mutant histone H3 plasmids. (C) Mutation of the tyrosine 99 phosphorylation site on H3 does not preclude the recognition of this histone by Rad53. After a 2 hour galactose (GAL) induction of HTH-tagged wild-type and mutant H3 in asynchronous cells carrying the RAD53-TAP allele, WCE were prepared. Rad53-TAP was immunoprecipitated (IPed) from the WCEs and the co-immunoprecipitated (co-IPed) HTH tagged H3 was detected by Western blotting with HA antibodies. The relative amount of endogenous and HTH tagged histone H3 present in the WCE is depicted in the lower panel to demonstrate that roughly equal amounts of material was used for each immunoprecipitation reaction.
    Figure Legend Snippet: Histones associated with Rad53 are phosphorylated and this modification is required for efficient histone degradation (A) Histone H3 associated with Rad53 is phosphorylated. TAP-tagged Rad53 was immunoprecipitated using IgG-Sepharose beads as described in the Methods section. Rad53-TAP bound to beads was first equilibrated with lysis buffer lacking phosphatase inhibitors and then incubated with or without 1mM ATP for 1hour at 30°C, following which the ATP treated sample was divided into three equal aliquots. One aliquot was left untreated; the second was incubated with λ-phosphatase (λ-PPase), while the third was treated with YOP tyrosine specific phosphatase (Y-PPase) for an additional hour at 30°C. All samples were boiled in SDS-PAGE sample loading buffer and proteins were resolved through a SDS 4–12% polyacrylamide gradient gel, which was processed for Western blotting with the H3-C antibody 9 . The migration of histone H3 present in whole cell extract (WCE) is indicated by the arrow. The lane labeled “No Tag” shows a mock immunoprecipitation with IgG-Sepharose performed on a strain where Rad53 was untagged. (B) Mutation of the tyrosine 99 phosphorylation site in histone H3 abolishes its degradation. Site-directed mutagenesis was used to mutate both the potential tyrosine (Y) phosphorylation sites in the H3 protein at positions 41 and 99 to phenylalanines (F), either individually (Y41F and Y99F), or in combination (Y41,99F). Histone degradation assays were carried out as described in the Methods section in wild type W303 cells bearing HTH-tagged wild type (WT) or mutant histone H3 plasmids. (C) Mutation of the tyrosine 99 phosphorylation site on H3 does not preclude the recognition of this histone by Rad53. After a 2 hour galactose (GAL) induction of HTH-tagged wild-type and mutant H3 in asynchronous cells carrying the RAD53-TAP allele, WCE were prepared. Rad53-TAP was immunoprecipitated (IPed) from the WCEs and the co-immunoprecipitated (co-IPed) HTH tagged H3 was detected by Western blotting with HA antibodies. The relative amount of endogenous and HTH tagged histone H3 present in the WCE is depicted in the lower panel to demonstrate that roughly equal amounts of material was used for each immunoprecipitation reaction.

    Techniques Used: Modification, Immunoprecipitation, Lysis, Incubation, SDS Page, Western Blot, Migration, Labeling, Mutagenesis

    12) Product Images from "A screening method for phosphohistidine phosphatase 1 activity"

    Article Title: A screening method for phosphohistidine phosphatase 1 activity

    Journal: Upsala Journal of Medical Sciences

    doi: 10.3109/03009734.2011.585253

    Dephosphorylation of purified and recombinant histone H4. Histone H4 was, after phosphorylation with phosphoramidate to 1.29 mol/mol, dephosphorylated with 2 pmoles PHPT1 for indicated times. The reaction was performed at 30°C and was interrupted by centrifugation of 50 μL of the reaction mixture through a spin column containing 210 μL DEAE-Sephacel equilibrated in 25 mM Tris/HCl, pH 8.5 at indicated times. Each time point was analysed in duplicate. The phosphate in the final eluate was determined by malachite reagent and histone by absorbance at 280 nm. Details are given in Material and methods.(◊) 40 μM purified phosphohistone H4, and (▪) 20 μM recombinant phosphohistone H4.
    Figure Legend Snippet: Dephosphorylation of purified and recombinant histone H4. Histone H4 was, after phosphorylation with phosphoramidate to 1.29 mol/mol, dephosphorylated with 2 pmoles PHPT1 for indicated times. The reaction was performed at 30°C and was interrupted by centrifugation of 50 μL of the reaction mixture through a spin column containing 210 μL DEAE-Sephacel equilibrated in 25 mM Tris/HCl, pH 8.5 at indicated times. Each time point was analysed in duplicate. The phosphate in the final eluate was determined by malachite reagent and histone by absorbance at 280 nm. Details are given in Material and methods.(◊) 40 μM purified phosphohistone H4, and (▪) 20 μM recombinant phosphohistone H4.

    Techniques Used: De-Phosphorylation Assay, Purification, Recombinant, Centrifugation

    13) Product Images from "Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum"

    Article Title: Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005686

    Histones and DNA contribute to the bactericidal activity of pea root border cells. (A) Histone H4, a component of the root cap secretome, is bactericidal to R . solanacearum . Percent live bacteria in the presence of histone H4 was determined by the BacLight LIVE/DEAD staining kit. A standard curve of percentage of live cells relative to SYTO9/PI fluorescence intensity was constructed using known ratios of live/dead bacteria (R 2 = 0.99). The experiment was repeated twice, each with three technical replicates. (B) Bactericidal activity of root border cells on R . solanacearum . This effect was blocked by addition of either anti-Histone H4 antibody or DNase I. Asterisks indicate treatment significantly different from the bacteria-only control (one-way ANOVA, **** p
    Figure Legend Snippet: Histones and DNA contribute to the bactericidal activity of pea root border cells. (A) Histone H4, a component of the root cap secretome, is bactericidal to R . solanacearum . Percent live bacteria in the presence of histone H4 was determined by the BacLight LIVE/DEAD staining kit. A standard curve of percentage of live cells relative to SYTO9/PI fluorescence intensity was constructed using known ratios of live/dead bacteria (R 2 = 0.99). The experiment was repeated twice, each with three technical replicates. (B) Bactericidal activity of root border cells on R . solanacearum . This effect was blocked by addition of either anti-Histone H4 antibody or DNase I. Asterisks indicate treatment significantly different from the bacteria-only control (one-way ANOVA, **** p

    Techniques Used: Activity Assay, Staining, Fluorescence, Construct

    14) Product Images from "Target identification reveals protein arginine methyltransferase 1 is a potential target of phenyl vinyl sulfone and its derivatives"

    Article Title: Target identification reveals protein arginine methyltransferase 1 is a potential target of phenyl vinyl sulfone and its derivatives

    Journal: Bioscience Reports

    doi: 10.1042/BSR20171717

    Effects of PVS, PVSN, Bay 11-7082, and AMI-1 on the in vitro enzyme activity of PRMT1 ( A ) and anti-PVS detection of PVS, PVSN, and Bay 11-7082 tagging of PRMT1 ( B ) The reaction mixture contained 100 ng of recombinant PRMT1, 1 µg of full-length recombinant Histone H4, 1 µM S-adenosylmethionine, various concentrations of PVS, PVSN, Bay 11-7082, or AMI-1 (AMI) in a total volume of 100 µl in PBS, pH 7.4. DMSO was used in the control. The reaction was incubated at 37°C for 30 min and 10 µl of the reaction product was examined by SDS/PAGE and immunoblotting with anti-H4R3me2a for the recognition of Histone H4 asymmetric dimethylation at Arg3 ( A ) and anti-PVS ( B ). The 0 µM groups were treated with the same volume of DMSO.
    Figure Legend Snippet: Effects of PVS, PVSN, Bay 11-7082, and AMI-1 on the in vitro enzyme activity of PRMT1 ( A ) and anti-PVS detection of PVS, PVSN, and Bay 11-7082 tagging of PRMT1 ( B ) The reaction mixture contained 100 ng of recombinant PRMT1, 1 µg of full-length recombinant Histone H4, 1 µM S-adenosylmethionine, various concentrations of PVS, PVSN, Bay 11-7082, or AMI-1 (AMI) in a total volume of 100 µl in PBS, pH 7.4. DMSO was used in the control. The reaction was incubated at 37°C for 30 min and 10 µl of the reaction product was examined by SDS/PAGE and immunoblotting with anti-H4R3me2a for the recognition of Histone H4 asymmetric dimethylation at Arg3 ( A ) and anti-PVS ( B ). The 0 µM groups were treated with the same volume of DMSO.

    Techniques Used: In Vitro, Activity Assay, Recombinant, Incubation, SDS Page

    15) Product Images from "Neutrophil dysregulation is pathogenic in idiopathic inflammatory myopathies"

    Article Title: Neutrophil dysregulation is pathogenic in idiopathic inflammatory myopathies

    Journal: JCI Insight

    doi: 10.1172/jci.insight.134189

    NETs interfere with myotube viability in a citrullinated histone-dependent manner. ( A ) Representative microphotographs display myotube fibers stained with DAPI and MF-20. Images were obtained at 48 hours of culture. Original magnification, ×20. ( B ) Percentage change in skeletal myotube cell area relative to that in untreated cells. Results represent individual measurements of 5 fields over 4 replicates of each experiment. Each condition included 4 independent wells and 5 images/well. Comparisons between groups were performed using a 2-sided Student’s t test. * P
    Figure Legend Snippet: NETs interfere with myotube viability in a citrullinated histone-dependent manner. ( A ) Representative microphotographs display myotube fibers stained with DAPI and MF-20. Images were obtained at 48 hours of culture. Original magnification, ×20. ( B ) Percentage change in skeletal myotube cell area relative to that in untreated cells. Results represent individual measurements of 5 fields over 4 replicates of each experiment. Each condition included 4 independent wells and 5 images/well. Comparisons between groups were performed using a 2-sided Student’s t test. * P

    Techniques Used: Staining

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    Positive Control:

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    Methylation:

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    Mutagenesis:

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    Isolation:

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    Article Snippet: .. For reactions, 2 μg of histones (isolated from HEK293T cells), recombinant histone 4 (New England Biolabs), synthetic histone 4 aa 1–21 and aa 15–24 peptides (Cayman Chemical), or synthetic histone H4 aa 1–21 with a K20A mutation (GenScript) were incubated with recombinant WT SMYD2 (Sigma-Aldrich) or SMYD2 Y240F (Active Motif) in a buffer containing 50 mM Tris-HCl, pH 9, 0.01% Tween 20, 2 mM DTT and 1.1 μCi of H3 -labeled SAM (Perkin Elmer) overnight at 30°C. .. Reaction mixtures were fractionated on 15% SDS-PAGE for proteins or on 10–20% Tris-Tricine gradient gels for peptides (BioRad).

    Labeling:

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    Article Snippet: .. For reactions, 2 μg of histones (isolated from HEK293T cells), recombinant histone 4 (New England Biolabs), synthetic histone 4 aa 1–21 and aa 15–24 peptides (Cayman Chemical), or synthetic histone H4 aa 1–21 with a K20A mutation (GenScript) were incubated with recombinant WT SMYD2 (Sigma-Aldrich) or SMYD2 Y240F (Active Motif) in a buffer containing 50 mM Tris-HCl, pH 9, 0.01% Tween 20, 2 mM DTT and 1.1 μCi of H3 -labeled SAM (Perkin Elmer) overnight at 30°C. .. Reaction mixtures were fractionated on 15% SDS-PAGE for proteins or on 10–20% Tris-Tricine gradient gels for peptides (BioRad).

    Purification:

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    Incubation:

    Article Title: UFL1 promotes histone H4 ufmylation and ATM activation
    Article Snippet: .. The purified proteins UFM1ΔC2, UBA5, UFC1, MBP-Ufl1, and histone H4, H3, H2A, or H2B (NEB) or nucleosome (Active motif)) were added into the reaction buffer containing 5 mM ATP and 10 mM MgCl2 and then incubated at 30 °C for 90 min. SDS sample buffer containing 5% β-mercaptoethanol was added into the mixture to stop the reaction. .. Immunofluorescence staining Cells were seeded on coverslips and then fixed with 3% paraformaldehyde on ice at indicated timepoints after IR treatment, washed with PBS, and permeabilized for 10 min with 0.5% Triton X-100.

    Article Title: SMYD2-Mediated Histone Methylation Contributes to HIV-1 Latency
    Article Snippet: .. For reactions, 2 μg of histones (isolated from HEK293T cells), recombinant histone 4 (New England Biolabs), synthetic histone 4 aa 1–21 and aa 15–24 peptides (Cayman Chemical), or synthetic histone H4 aa 1–21 with a K20A mutation (GenScript) were incubated with recombinant WT SMYD2 (Sigma-Aldrich) or SMYD2 Y240F (Active Motif) in a buffer containing 50 mM Tris-HCl, pH 9, 0.01% Tween 20, 2 mM DTT and 1.1 μCi of H3 -labeled SAM (Perkin Elmer) overnight at 30°C. .. Reaction mixtures were fractionated on 15% SDS-PAGE for proteins or on 10–20% Tris-Tricine gradient gels for peptides (BioRad).

    Activity Assay:

    Article Title: PRMT7 regulates RNA-binding capacity and protein stability in Leishmania parasites
    Article Snippet: .. Reactions were then halted by the addition of 2× SDS sample buffer (125 mM Tris–Cl pH 6.8, 20% glycerol, 2% SDS, 0.7 M β-mercaptoethanol and 0.01% bromophenol blue) at 95°C for 5 min. Human histone H4 was used as a positive control for PRMT7 activity (1 μg per reaction; New England Biolabs). ..

    Recombinant:

    Article Title: SMYD2-Mediated Histone Methylation Contributes to HIV-1 Latency
    Article Snippet: .. For reactions, 2 μg of histones (isolated from HEK293T cells), recombinant histone 4 (New England Biolabs), synthetic histone 4 aa 1–21 and aa 15–24 peptides (Cayman Chemical), or synthetic histone H4 aa 1–21 with a K20A mutation (GenScript) were incubated with recombinant WT SMYD2 (Sigma-Aldrich) or SMYD2 Y240F (Active Motif) in a buffer containing 50 mM Tris-HCl, pH 9, 0.01% Tween 20, 2 mM DTT and 1.1 μCi of H3 -labeled SAM (Perkin Elmer) overnight at 30°C. .. Reaction mixtures were fractionated on 15% SDS-PAGE for proteins or on 10–20% Tris-Tricine gradient gels for peptides (BioRad).

    Article Title: Nuclear Cyclin D1/CDK4 Kinase Regulates CUL4 Expression and Triggers Neoplastic Growth via Activation of the PRMT5 Methyltransferase
    Article Snippet: .. The methylation reaction included PRMT5 immune complexes on beads, 1μg recombinant Histone H4 (NEB), and 2.75μCi S-adenosyl-L-(methyl-3 H)methionine (Amersham Pharmacia) in a total volume of 25μl for 1.5 hours at 30°C. .. The reaction mixture was resolved on a SDS-polyacrylamide gel, and modified histone H4 was detected by fluorography.

    Article Title: Dual effect of histone H4 on prothrombin activation
    Article Snippet: .. Recombinant histone H4 was purchased from New England BioLabs. .. Activation of prothrombin (0.1 mg/mL, 1.4 μM) was carried out at room temperature for 5 min as reported elsewhere [ , ] in the presence of different concentrations (0–10 μM) of histone H4 under experimental conditions of 145 mM NaCl, 5 mM CaCl2 , 10 mM Tris, pH 7.4.

    Article Title: Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis
    Article Snippet: .. Recombinant histone H4 was purchased from NEB (Cat#: M2504S). .. For the in vitro acetylation assay, 1 μg of recombinant histone H4 was incubated with 250 ng of purified Esco2-Flag or Esco2-W530G-Flag in 50 μl of acetyltransferase assay buffer (50 mM Tris–HCl pH8.0, 10% glycerol, 10 mM butyric acid, 0.1 mM ethylenediaminetetraacetic acid, 1 mM Dithiothreitol (DTT) and 1mM Phenylmethylsulfonyl fluoride (PMSF) with or without 10 μM Ac-CoA (Sigma) at 30°C for 1 h on a rotating platform.

    Article Title: Pressure-assisted sample preparation for proteomic analysis
    Article Snippet: .. Recombinant human histone H4 ( ) was purchased from New England Biolabs (Ipswich, MA, USA). .. Primary human monocytes, obtained by elutriation from healthy donors as described in Refs. [ , ], were used to isolate histone fractions using a Qproteome Nuclear Protein Kit (Qiagen, Hilden, Germany).

    Article Title: Tousled kinase TLK1B counteracts the effect of Asf1 in inhibition of histone H3-H4 tetramer formation
    Article Snippet: .. Materials and methods Recombinant human histone H4 was purchased from NE Biolabs and bovine H3 from Roche. .. Recombinant human TLK1B and Asf1B were prepared as described previously [ ].

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    New England Biolabs recombinant histone 4
    SMYD2 methylates <t>histone</t> 4 at lysine 20 In vitro methylation assays, including histones isolated from HEK293T, recombinant full-length histone H4 or two short synthetic histone H4 peptides (aa 1–21 and aa 15–24) that were incubated with recombinant SMYD2 enzyme and radiolabeled H 3 -S-adenosyl-L-methionine (SAM) in the presence or absence of AZ391. Reactions were resolved by gel electrophoresis and developed by autoradiography. (a) In vitro methylation assays of histones isolated from HEK293T cells. (b) In vitro SMYD2 methylation assay of recombinant full-length histone H4, with or without AZ391. (c) In vitro SMYD2 methylation assays of synthetic histone H4 peptides (aa 1–21, left, and aa 15–24, right) in the presence or absence of AZ391. (d) In vitro SMYD2 methylation assay of synthetic histone H4 peptide (aa 1–21) with or without a K20A mutation. (e) In vitro methylation assays of human recombinant histone H4 using wildtype or catalytically inactive (Y240F) SMYD2. All in vitro methylation assays of recombinant histone H4 or H4 peptides were repeated at least three times, and representative Coomassie stain (left) and autoradiography (right) are shown. (f–h) In vitro SMYD2 methylation assay of recombinant full-length histone H4 was subjected to mass spectrometry. (f) Annotated HCD MS/MS spectrum of the histone H4 LysC peptide RHRKmeVLRDIQGITK containing K20 methylation. Blue lines indicate b ions and purple lines indicate y ions, with specific ions labeled atop each peak. (g–h) Integrated MS1 intensity for the RHRKmeVLRDIQGITK peptide (g) and an unmodified histone H4 peptide (h) across different samples. Error bars indicate standard deviation between technical replicate MS analyses.
    Recombinant Histone 4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SMYD2 methylates histone 4 at lysine 20 In vitro methylation assays, including histones isolated from HEK293T, recombinant full-length histone H4 or two short synthetic histone H4 peptides (aa 1–21 and aa 15–24) that were incubated with recombinant SMYD2 enzyme and radiolabeled H 3 -S-adenosyl-L-methionine (SAM) in the presence or absence of AZ391. Reactions were resolved by gel electrophoresis and developed by autoradiography. (a) In vitro methylation assays of histones isolated from HEK293T cells. (b) In vitro SMYD2 methylation assay of recombinant full-length histone H4, with or without AZ391. (c) In vitro SMYD2 methylation assays of synthetic histone H4 peptides (aa 1–21, left, and aa 15–24, right) in the presence or absence of AZ391. (d) In vitro SMYD2 methylation assay of synthetic histone H4 peptide (aa 1–21) with or without a K20A mutation. (e) In vitro methylation assays of human recombinant histone H4 using wildtype or catalytically inactive (Y240F) SMYD2. All in vitro methylation assays of recombinant histone H4 or H4 peptides were repeated at least three times, and representative Coomassie stain (left) and autoradiography (right) are shown. (f–h) In vitro SMYD2 methylation assay of recombinant full-length histone H4 was subjected to mass spectrometry. (f) Annotated HCD MS/MS spectrum of the histone H4 LysC peptide RHRKmeVLRDIQGITK containing K20 methylation. Blue lines indicate b ions and purple lines indicate y ions, with specific ions labeled atop each peak. (g–h) Integrated MS1 intensity for the RHRKmeVLRDIQGITK peptide (g) and an unmodified histone H4 peptide (h) across different samples. Error bars indicate standard deviation between technical replicate MS analyses.

    Journal: Cell host & microbe

    Article Title: SMYD2-Mediated Histone Methylation Contributes to HIV-1 Latency

    doi: 10.1016/j.chom.2017.04.011

    Figure Lengend Snippet: SMYD2 methylates histone 4 at lysine 20 In vitro methylation assays, including histones isolated from HEK293T, recombinant full-length histone H4 or two short synthetic histone H4 peptides (aa 1–21 and aa 15–24) that were incubated with recombinant SMYD2 enzyme and radiolabeled H 3 -S-adenosyl-L-methionine (SAM) in the presence or absence of AZ391. Reactions were resolved by gel electrophoresis and developed by autoradiography. (a) In vitro methylation assays of histones isolated from HEK293T cells. (b) In vitro SMYD2 methylation assay of recombinant full-length histone H4, with or without AZ391. (c) In vitro SMYD2 methylation assays of synthetic histone H4 peptides (aa 1–21, left, and aa 15–24, right) in the presence or absence of AZ391. (d) In vitro SMYD2 methylation assay of synthetic histone H4 peptide (aa 1–21) with or without a K20A mutation. (e) In vitro methylation assays of human recombinant histone H4 using wildtype or catalytically inactive (Y240F) SMYD2. All in vitro methylation assays of recombinant histone H4 or H4 peptides were repeated at least three times, and representative Coomassie stain (left) and autoradiography (right) are shown. (f–h) In vitro SMYD2 methylation assay of recombinant full-length histone H4 was subjected to mass spectrometry. (f) Annotated HCD MS/MS spectrum of the histone H4 LysC peptide RHRKmeVLRDIQGITK containing K20 methylation. Blue lines indicate b ions and purple lines indicate y ions, with specific ions labeled atop each peak. (g–h) Integrated MS1 intensity for the RHRKmeVLRDIQGITK peptide (g) and an unmodified histone H4 peptide (h) across different samples. Error bars indicate standard deviation between technical replicate MS analyses.

    Article Snippet: For reactions, 2 μg of histones (isolated from HEK293T cells), recombinant histone 4 (New England Biolabs), synthetic histone 4 aa 1–21 and aa 15–24 peptides (Cayman Chemical), or synthetic histone H4 aa 1–21 with a K20A mutation (GenScript) were incubated with recombinant WT SMYD2 (Sigma-Aldrich) or SMYD2 Y240F (Active Motif) in a buffer containing 50 mM Tris-HCl, pH 9, 0.01% Tween 20, 2 mM DTT and 1.1 μCi of H3 -labeled SAM (Perkin Elmer) overnight at 30°C.

    Techniques: In Vitro, Methylation, Isolation, Recombinant, Incubation, Nucleic Acid Electrophoresis, Autoradiography, Mutagenesis, Staining, Mass Spectrometry, Labeling, Standard Deviation

    Time optimization at 15 kpsi for 30, 120, and 200 min for two peptides from the N-terminal tail portion of recombinant human histone H4.

    Journal: Analytical biochemistry

    Article Title: Pressure-assisted sample preparation for proteomic analysis

    doi: 10.1016/j.ab.2013.03.023

    Figure Lengend Snippet: Time optimization at 15 kpsi for 30, 120, and 200 min for two peptides from the N-terminal tail portion of recombinant human histone H4.

    Article Snippet: Recombinant human histone H4 ( ) was purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques: Recombinant

    MALDI–TOF mass spectrum of recombinant human histone H4 digested using the PCT method. The numbered peaks match those generated by conventional chymotrypsin digestion, providing 95% sequence coverage, and are listed in .

    Journal: Analytical biochemistry

    Article Title: Pressure-assisted sample preparation for proteomic analysis

    doi: 10.1016/j.ab.2013.03.023

    Figure Lengend Snippet: MALDI–TOF mass spectrum of recombinant human histone H4 digested using the PCT method. The numbered peaks match those generated by conventional chymotrypsin digestion, providing 95% sequence coverage, and are listed in .

    Article Snippet: Recombinant human histone H4 ( ) was purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques: Recombinant, Generated, Sequencing

    Pressure optimization at 30 min for 10, 15, and 25 kpsi for two peptides from the N-terminal tail portion of recombinant human histone H4.

    Journal: Analytical biochemistry

    Article Title: Pressure-assisted sample preparation for proteomic analysis

    doi: 10.1016/j.ab.2013.03.023

    Figure Lengend Snippet: Pressure optimization at 30 min for 10, 15, and 25 kpsi for two peptides from the N-terminal tail portion of recombinant human histone H4.

    Article Snippet: Recombinant human histone H4 ( ) was purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques: Recombinant

    RP–HPLC chromatogram showing fractionation of histones using UV detector set at 214 nm. The inset shows an enlarged range of the peptides containing peaks assigned to respective histones.

    Journal: Analytical biochemistry

    Article Title: Pressure-assisted sample preparation for proteomic analysis

    doi: 10.1016/j.ab.2013.03.023

    Figure Lengend Snippet: RP–HPLC chromatogram showing fractionation of histones using UV detector set at 214 nm. The inset shows an enlarged range of the peptides containing peaks assigned to respective histones.

    Article Snippet: Recombinant human histone H4 ( ) was purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques: High Performance Liquid Chromatography, Fractionation

    Chymotryptic digestion of native histone H4 using conventional and PCT methods

    Journal: Analytical biochemistry

    Article Title: Pressure-assisted sample preparation for proteomic analysis

    doi: 10.1016/j.ab.2013.03.023

    Figure Lengend Snippet: Chymotryptic digestion of native histone H4 using conventional and PCT methods

    Article Snippet: Recombinant human histone H4 ( ) was purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques:

    Ufmylation of histone H4 enhances trimethylation of H3K9 at DSB. a , b Analysis of H3K9 trimethylation and Suv39H1 recruitment by Chromatin IP (ChIP) at DSB sites from MDA-MB-231 ROS8 cells with indicated treatments. The y -axis represents relative enrichment of target protein binding DNA compared to input. Target sequence of UFL1 shRNA (not inducible shRNA) in a is the same as inducible sh2. The data presented are mean ± s.e.m. for n = 3 independent experiments. ** p

    Journal: Nature Communications

    Article Title: UFL1 promotes histone H4 ufmylation and ATM activation

    doi: 10.1038/s41467-019-09175-0

    Figure Lengend Snippet: Ufmylation of histone H4 enhances trimethylation of H3K9 at DSB. a , b Analysis of H3K9 trimethylation and Suv39H1 recruitment by Chromatin IP (ChIP) at DSB sites from MDA-MB-231 ROS8 cells with indicated treatments. The y -axis represents relative enrichment of target protein binding DNA compared to input. Target sequence of UFL1 shRNA (not inducible shRNA) in a is the same as inducible sh2. The data presented are mean ± s.e.m. for n = 3 independent experiments. ** p

    Article Snippet: The purified proteins UFM1ΔC2, UBA5, UFC1, MBP-Ufl1, and histone H4, H3, H2A, or H2B (NEB) or nucleosome (Active motif)) were added into the reaction buffer containing 5 mM ATP and 10 mM MgCl2 and then incubated at 30 °C for 90 min. SDS sample buffer containing 5% β-mercaptoethanol was added into the mixture to stop the reaction.

    Techniques: Chromatin Immunoprecipitation, Multiple Displacement Amplification, Protein Binding, Sequencing, shRNA

    Model for ATM activation by UFL1. When DNA damage occurs, UFL1 is recruited by MRN complex and monoufmylates histone H4, thus recruiting SUV39H1 to DSBs to trimethylate H3K9 thereby forming H3K9me3. Tip60 binds to H3K9me3, acetylates ATM, and promotes ATM activation. C-Abl also phosphorylates Tip60 and enhances Tip60 acetyltransferase activity. Activated ATM phosphorylates UFL1 at Ser462, and enhances its activity thereby amplifying ATM activation signal and forming a positive feedback loop

    Journal: Nature Communications

    Article Title: UFL1 promotes histone H4 ufmylation and ATM activation

    doi: 10.1038/s41467-019-09175-0

    Figure Lengend Snippet: Model for ATM activation by UFL1. When DNA damage occurs, UFL1 is recruited by MRN complex and monoufmylates histone H4, thus recruiting SUV39H1 to DSBs to trimethylate H3K9 thereby forming H3K9me3. Tip60 binds to H3K9me3, acetylates ATM, and promotes ATM activation. C-Abl also phosphorylates Tip60 and enhances Tip60 acetyltransferase activity. Activated ATM phosphorylates UFL1 at Ser462, and enhances its activity thereby amplifying ATM activation signal and forming a positive feedback loop

    Article Snippet: The purified proteins UFM1ΔC2, UBA5, UFC1, MBP-Ufl1, and histone H4, H3, H2A, or H2B (NEB) or nucleosome (Active motif)) were added into the reaction buffer containing 5 mM ATP and 10 mM MgCl2 and then incubated at 30 °C for 90 min. SDS sample buffer containing 5% β-mercaptoethanol was added into the mixture to stop the reaction.

    Techniques: Activation Assay, Activity Assay

    UFL1 monoufmylates histone H4 and promotes ATM activation. a Selected proteins identified by mass spectrometry from irradiated 293T cell expressing Flag-His vector or Flag-His-UFM1. N = 1 sample in each group was analyzed. The full list of identified proteins is provided in Supplementary Data 1 . Among histone proteins, only H4 is enriched in the Flag-His-UFM1 purification (9 unique/20 total peptides) compared to the Flag-His purification (6 unique/7 total peptides), suggesting that H4 might be ufmylated. b Flag-His-ufmylated proteins were purified from 293T cells before and after IR (2 Gy) after purification with nickel beads and anti-Flag agarose. The immunoprecipitates were detected with indicated antibodies. c Flag-His-ufmylated H4 was purified from control and UFL1 knockdown cells with or without 2 Gy IR and blotted with indicated antibodies. d In vitro ufmylation assay. Purified UBA5, UFC1, UFL1, UFM1, and H4 proteins were incubated together in the presence of ATP and MgCl 2 at 30 ° C for 90 min. The reaction products were probed with indicated antibodies. e Wildtype (WT) histone H4 and 11 different single lysine (K) to arginine (R) mutants were transfected into U2OS cells. Flag and His tandem purification was performed and H4 ufmylation was analyzed. f Constructs expressing WT or K31R H4 were transfected into U2OS tet-on UFL1 shRNA expressing cells, and the cells were treated with doxycycline as indicated. Thirty minutes after 2 Gy IR, the cells were harvested and blotted with indicated antibodies. g Colony formation of U2OS cells expressing WT H4 or H4K31R following IR. The data presented are mean ± s.e.m. for n = 3 independent experiments. Statistical significance was calculated using two-way ANOVA. * p

    Journal: Nature Communications

    Article Title: UFL1 promotes histone H4 ufmylation and ATM activation

    doi: 10.1038/s41467-019-09175-0

    Figure Lengend Snippet: UFL1 monoufmylates histone H4 and promotes ATM activation. a Selected proteins identified by mass spectrometry from irradiated 293T cell expressing Flag-His vector or Flag-His-UFM1. N = 1 sample in each group was analyzed. The full list of identified proteins is provided in Supplementary Data 1 . Among histone proteins, only H4 is enriched in the Flag-His-UFM1 purification (9 unique/20 total peptides) compared to the Flag-His purification (6 unique/7 total peptides), suggesting that H4 might be ufmylated. b Flag-His-ufmylated proteins were purified from 293T cells before and after IR (2 Gy) after purification with nickel beads and anti-Flag agarose. The immunoprecipitates were detected with indicated antibodies. c Flag-His-ufmylated H4 was purified from control and UFL1 knockdown cells with or without 2 Gy IR and blotted with indicated antibodies. d In vitro ufmylation assay. Purified UBA5, UFC1, UFL1, UFM1, and H4 proteins were incubated together in the presence of ATP and MgCl 2 at 30 ° C for 90 min. The reaction products were probed with indicated antibodies. e Wildtype (WT) histone H4 and 11 different single lysine (K) to arginine (R) mutants were transfected into U2OS cells. Flag and His tandem purification was performed and H4 ufmylation was analyzed. f Constructs expressing WT or K31R H4 were transfected into U2OS tet-on UFL1 shRNA expressing cells, and the cells were treated with doxycycline as indicated. Thirty minutes after 2 Gy IR, the cells were harvested and blotted with indicated antibodies. g Colony formation of U2OS cells expressing WT H4 or H4K31R following IR. The data presented are mean ± s.e.m. for n = 3 independent experiments. Statistical significance was calculated using two-way ANOVA. * p

    Article Snippet: The purified proteins UFM1ΔC2, UBA5, UFC1, MBP-Ufl1, and histone H4, H3, H2A, or H2B (NEB) or nucleosome (Active motif)) were added into the reaction buffer containing 5 mM ATP and 10 mM MgCl2 and then incubated at 30 °C for 90 min. SDS sample buffer containing 5% β-mercaptoethanol was added into the mixture to stop the reaction.

    Techniques: Activation Assay, Mass Spectrometry, Irradiation, Expressing, Plasmid Preparation, Purification, In Vitro, Incubation, Transfection, Construct, shRNA

    Effect of histone H4 on prothrombin activation by factor Xa

    Journal: Journal of thrombosis and haemostasis : JTH

    Article Title: Dual effect of histone H4 on prothrombin activation

    doi: 10.1111/jth.13400

    Figure Lengend Snippet: Effect of histone H4 on prothrombin activation by factor Xa

    Article Snippet: Recombinant histone H4 was purchased from New England BioLabs.

    Techniques: Activation Assay