histone h2a  (New England Biolabs)


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    Name:
    Histone H2A Human Recombinant
    Description:
    Histone H2A Human Recombinant 100 ug
    Catalog Number:
    m2502s
    Price:
    82
    Size:
    100 ug
    Category:
    DNA Binding Proteins
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    New England Biolabs histone h2a
    Histone H2A Human Recombinant
    Histone H2A Human Recombinant 100 ug
    https://www.bioz.com/result/histone h2a/product/New England Biolabs
    Average 93 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    histone h2a - by Bioz Stars, 2020-08
    93/100 stars

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    1) Product Images from "Breaching peripheral tolerance promotes the production of HIV-1–neutralizing antibodies"

    Article Title: Breaching peripheral tolerance promotes the production of HIV-1–neutralizing antibodies

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20161190

    Histone H2A-reactive IgM monoclonal antibodies, isolated from B6.Sle123 mice, neutralize tier 2 strains of HIV-1. Hybridomas were generated from splenocytes isolated from B6.Sle123 mice that displayed serum tier 2 HIV-1 neutralization ( n = 2). (A) Purified monoclonal IgM and IgG antibodies ( n = 8) were tested for HIV-1 neutralization against 4 strains of HIV-1 and reported as IC 50 (the concentration of antibody required for 50% neutralization). (B) Specificities of the monoclonal antibodies were tested using ELISA against the histone H2A, histone H2B, and chromatin nuclear antigens in addition to HIV-1 gp140 (YU2) Env and the CD4bs (RSC3) epitope, as well as a CD4bs-negative control (ΔRSC3). The two neutralizing IgM mAbs (P4E4 and O4C5) are shown with red symbols, and lines and non-neutralizing mAbs shown with solid black symbols and lines for IgM and gray symbols and dashed lines for IgG. IgM and IgG control antibodies were used to determine the background of the assay and for which ODs above this line were considered positive. Data are representative from three independent experiments.
    Figure Legend Snippet: Histone H2A-reactive IgM monoclonal antibodies, isolated from B6.Sle123 mice, neutralize tier 2 strains of HIV-1. Hybridomas were generated from splenocytes isolated from B6.Sle123 mice that displayed serum tier 2 HIV-1 neutralization ( n = 2). (A) Purified monoclonal IgM and IgG antibodies ( n = 8) were tested for HIV-1 neutralization against 4 strains of HIV-1 and reported as IC 50 (the concentration of antibody required for 50% neutralization). (B) Specificities of the monoclonal antibodies were tested using ELISA against the histone H2A, histone H2B, and chromatin nuclear antigens in addition to HIV-1 gp140 (YU2) Env and the CD4bs (RSC3) epitope, as well as a CD4bs-negative control (ΔRSC3). The two neutralizing IgM mAbs (P4E4 and O4C5) are shown with red symbols, and lines and non-neutralizing mAbs shown with solid black symbols and lines for IgM and gray symbols and dashed lines for IgG. IgM and IgG control antibodies were used to determine the background of the assay and for which ODs above this line were considered positive. Data are representative from three independent experiments.

    Techniques Used: Isolation, Mouse Assay, Generated, Neutralization, Purification, Concentration Assay, Enzyme-linked Immunosorbent Assay, Negative Control

    Elevated IgM anti-histone H2A titers correlate with tier 2 HIV-1 neutralization by pristane treated wild-type C57BL/6 mice. Total serum concentrations of (A) IgM, (B) IgG, and (C) relative titers of serum IgM anti-H2A are shown for naive (open circles) B6 mice or B6 mice treated 30 d with pristane only (gray circles) and subsequently immunized 2X or 3X (black circles) with alum alone or Env + alum. Serum from individual B6 mice (regardless of treatment with pristane alone, alum alone, or Env + alum) were separated based on neutralization of ≥1 tier 2 HIV-1 strains and measured for (D) IgM anti-H2A or (E) IgM anti-H2B relative titers. Mice neutralizing only tier 1 strains were included in the tier 2 nonneutralizer group (mostly 3X). Each symbol represents measurements for one mouse, and all data are plotted as the arithmetic mean ± SEM. All P-values were calculated using Student’s t test assuming unequal variances. *, P
    Figure Legend Snippet: Elevated IgM anti-histone H2A titers correlate with tier 2 HIV-1 neutralization by pristane treated wild-type C57BL/6 mice. Total serum concentrations of (A) IgM, (B) IgG, and (C) relative titers of serum IgM anti-H2A are shown for naive (open circles) B6 mice or B6 mice treated 30 d with pristane only (gray circles) and subsequently immunized 2X or 3X (black circles) with alum alone or Env + alum. Serum from individual B6 mice (regardless of treatment with pristane alone, alum alone, or Env + alum) were separated based on neutralization of ≥1 tier 2 HIV-1 strains and measured for (D) IgM anti-H2A or (E) IgM anti-H2B relative titers. Mice neutralizing only tier 1 strains were included in the tier 2 nonneutralizer group (mostly 3X). Each symbol represents measurements for one mouse, and all data are plotted as the arithmetic mean ± SEM. All P-values were calculated using Student’s t test assuming unequal variances. *, P

    Techniques Used: Neutralization, Mouse Assay

    B6.Sle123 HIV-1 neutralizers harbor elevated levels of IgM anti-histone H2A. (A) Sera from B6 (open), B6.Sle123 nonneutralizers (gray), and neutralizers (black) were interrogated with an autoantigen array and results for the IgM reactive with the indicated anti-DNA antigens (top left), RNA-binding proteins (bottom left), and anti-histone antigens (top right), as well as IgG anti-histone antigens (bottom right) are shown. Relative serum titers of (B) IgM anti-H2A (left) and anti-H2B (right) measured by ELISA in B6.Sle123 neutralizers and nonneutralizers. Each symbol represents measurements for one mouse, and all data are plotted as the arithmetic mean ± SEM. P-values were calculated using the Mann-Whitney nonparametric test; *, P
    Figure Legend Snippet: B6.Sle123 HIV-1 neutralizers harbor elevated levels of IgM anti-histone H2A. (A) Sera from B6 (open), B6.Sle123 nonneutralizers (gray), and neutralizers (black) were interrogated with an autoantigen array and results for the IgM reactive with the indicated anti-DNA antigens (top left), RNA-binding proteins (bottom left), and anti-histone antigens (top right), as well as IgG anti-histone antigens (bottom right) are shown. Relative serum titers of (B) IgM anti-H2A (left) and anti-H2B (right) measured by ELISA in B6.Sle123 neutralizers and nonneutralizers. Each symbol represents measurements for one mouse, and all data are plotted as the arithmetic mean ± SEM. P-values were calculated using the Mann-Whitney nonparametric test; *, P

    Techniques Used: RNA Binding Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    2) Product Images from "Structure Change from β-Strand and Turn to α-Helix in Histone H2A-H2B Induced by DNA Damage Response"

    Article Title: Structure Change from β-Strand and Turn to α-Helix in Histone H2A-H2B Induced by DNA Damage Response

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2016.06.002

    ( a ) Temperature dependence of the CD intensity at 222 nm of H2A-H2B extracted from core histones in unirradiated (Unirrad., open circle ) and x-irradiated (Irrad., solid circle ) cells. Inset: magnification of the unirradiated sample between 320
    Figure Legend Snippet: ( a ) Temperature dependence of the CD intensity at 222 nm of H2A-H2B extracted from core histones in unirradiated (Unirrad., open circle ) and x-irradiated (Irrad., solid circle ) cells. Inset: magnification of the unirradiated sample between 320

    Techniques Used: Irradiation

    Thermodynamic parameters of H2A-H2B
    Figure Legend Snippet: Thermodynamic parameters of H2A-H2B

    Techniques Used:

    ( a–c ) CD spectra of H2A-H2B extracted from core histones in unirradiated (Unirrad., black ) and x-irradiated (Irrad., red ) cells, measured at ( a ) 294, ( b ) 310, and ( c ) 330 K. In ( a ), a CD spectrum of H2A-H2B irradiated with 40 Gy x-rays
    Figure Legend Snippet: ( a–c ) CD spectra of H2A-H2B extracted from core histones in unirradiated (Unirrad., black ) and x-irradiated (Irrad., red ) cells, measured at ( a ) 294, ( b ) 310, and ( c ) 330 K. In ( a ), a CD spectrum of H2A-H2B irradiated with 40 Gy x-rays

    Techniques Used: Irradiation

    ( a–d ) Temperature dependence of the contents of ( a ) α -helix, ( b ) β -strand, ( c ) turn, and ( d ) other structures of H2A-H2B extracted from core histones in unirradiated (Unirrad., open circle ) and x-irradiated (Irrad., solid
    Figure Legend Snippet: ( a–d ) Temperature dependence of the contents of ( a ) α -helix, ( b ) β -strand, ( c ) turn, and ( d ) other structures of H2A-H2B extracted from core histones in unirradiated (Unirrad., open circle ) and x-irradiated (Irrad., solid

    Techniques Used: Irradiation

    ( a ) SDS-PAGE analysis of H2A-H2B extracted from core histones in unirradiated cells and 40 Gy irradiated cells after 30 min incubation. The lane labeled M refers to the standard molecular weight marker (XL-Ladder (low range); APRO Life
    Figure Legend Snippet: ( a ) SDS-PAGE analysis of H2A-H2B extracted from core histones in unirradiated cells and 40 Gy irradiated cells after 30 min incubation. The lane labeled M refers to the standard molecular weight marker (XL-Ladder (low range); APRO Life

    Techniques Used: SDS Page, Irradiation, Incubation, Labeling, Molecular Weight, Marker

    3) Product Images from "Probing the catalytic functions of Bub1 kinase using the small molecule inhibitors BAY-320 and BAY-524"

    Article Title: Probing the catalytic functions of Bub1 kinase using the small molecule inhibitors BAY-320 and BAY-524

    Journal: eLife

    doi: 10.7554/eLife.12187

    BAY-320 and BAY-524 inhibit Bub1 kinase. ( A, B ) BAY-320 and BAY-524 treatment coordinately reduces histone H2A-T120 phosphorylation as well as Aurora B centromere/KT binding, until maximal Bub1 inhibition is reached at 10 μM. Asynchronous cultures of HeLa S3 (left panels) and RPE1 cells (right panels) were treated with the proteasomal inhibitor MG132 for 2 hr, followed by the addition of 3.3 μM nocodazole and increasing doses of BAY-320 ( A ) or BAY-524 ( B ) for 1 hr. The cells were fixed and analyzed by immunofluorescence microscopy (IFM). Scatter plots show centromere/KT levels of pT120-H2A and Aurora B (n = 19–28 cells per condition). Bars represent mean values. ( C ) Untreated HeLa cells (red) or HeLa cells treated with nocodazole for 16 hr, followed by various concentrations of BAY-320 (green) or solvent (black) for 1 hr, were fixed and analyzed by quantitative in-cell western. Plot shows total pT120-H2A signal intensity. Grey area highlights the concentration range between 3 and 10 μM. The IC 50 (reflecting the inhibition of Bub1 kinase activity compared to control and normalized to cell number) was determined to be 379 +/- 156 nM. DOI: http://dx.doi.org/10.7554/eLife.12187.004
    Figure Legend Snippet: BAY-320 and BAY-524 inhibit Bub1 kinase. ( A, B ) BAY-320 and BAY-524 treatment coordinately reduces histone H2A-T120 phosphorylation as well as Aurora B centromere/KT binding, until maximal Bub1 inhibition is reached at 10 μM. Asynchronous cultures of HeLa S3 (left panels) and RPE1 cells (right panels) were treated with the proteasomal inhibitor MG132 for 2 hr, followed by the addition of 3.3 μM nocodazole and increasing doses of BAY-320 ( A ) or BAY-524 ( B ) for 1 hr. The cells were fixed and analyzed by immunofluorescence microscopy (IFM). Scatter plots show centromere/KT levels of pT120-H2A and Aurora B (n = 19–28 cells per condition). Bars represent mean values. ( C ) Untreated HeLa cells (red) or HeLa cells treated with nocodazole for 16 hr, followed by various concentrations of BAY-320 (green) or solvent (black) for 1 hr, were fixed and analyzed by quantitative in-cell western. Plot shows total pT120-H2A signal intensity. Grey area highlights the concentration range between 3 and 10 μM. The IC 50 (reflecting the inhibition of Bub1 kinase activity compared to control and normalized to cell number) was determined to be 379 +/- 156 nM. DOI: http://dx.doi.org/10.7554/eLife.12187.004

    Techniques Used: Binding Assay, Inhibition, Immunofluorescence, Microscopy, In-Cell ELISA, Concentration Assay, Activity Assay

    4) Product Images from "Proteolytic Histone Modification by Mast Cell Tryptase, a Serglycin Proteoglycan-dependent Secretory Granule Protease *"

    Article Title: Proteolytic Histone Modification by Mast Cell Tryptase, a Serglycin Proteoglycan-dependent Secretory Granule Protease *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.546895

    The processing of histones in viable and apoptotic mast cells is dependent on tryptase and serglycin. WT, serglycin −/− , and mMCP-6 −/− mast cells were treated with cytotoxic agent (CHX) or left untreated. In addition, WT cells were incubated in the presence of both cytotoxic agent and a general serine protease inhibitor (Pefabloc) as indicated. At the time points indicated, total cell extracts (corresponding to 0.5 × 10 6 cells/sample) were prepared and subjected to immunoblot analysis for core histones H2A, H2B, H3, and H4. β-Actin was used as loading control.
    Figure Legend Snippet: The processing of histones in viable and apoptotic mast cells is dependent on tryptase and serglycin. WT, serglycin −/− , and mMCP-6 −/− mast cells were treated with cytotoxic agent (CHX) or left untreated. In addition, WT cells were incubated in the presence of both cytotoxic agent and a general serine protease inhibitor (Pefabloc) as indicated. At the time points indicated, total cell extracts (corresponding to 0.5 × 10 6 cells/sample) were prepared and subjected to immunoblot analysis for core histones H2A, H2B, H3, and H4. β-Actin was used as loading control.

    Techniques Used: Incubation, Protease Inhibitor

    5) Product Images from "Elevated H3K79 homocysteinylation causes abnormal gene expression during neural development and subsequent neural tube defects"

    Article Title: Elevated H3K79 homocysteinylation causes abnormal gene expression during neural development and subsequent neural tube defects

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05451-7

    Histone homocysteinylation is a common modification among different tissues and species. a A typical HPLC-MS/MS spectra of a tryptic peptide ‘RGGVK Hcy RISGLIYEETR’ harboring H4K44 homocystylation, derived from human brain. The x and y axes represent m / z and relative ion intensity, respectively. A series of b- and y-type homocysteinylation fragment ions are evident which not only provide reliable sequence information, but also indicate an unambiguous +174.04600 Da shift for Hcy. b Schematic illustration of homocysteinylation sites of histone lysine residues in human normal brain samples identified using HPLC-MS/MS. The red diamond shape depicts homocysteinylation sites in core histones (H3, H4, H2a, and H2b). The number underneath each red lysine residue (K) represents the position of the particular lysine residue within each respective histone. c Verification of anti-KHcy antibody. The homocysteinylation levels of BSA (Bovine Serum Album) and KHcy modified BSA were detected with anti-KHcy antibody under the presence of 0, 2, or 5 µg/ml of Hcy modified lysine (KHcy as competitor). CBB Coomassie Brilliant Blue staining. These test repeated for 3 times and the quantitation of the western blotting showed on right. In the BSA group, the relative K-Hcy levels were 1 ± 0.05, 1.15 ± 0.10; 1.11 ± 0.78. In the BSA-Hcy group, the relative K-Hcy levels were 10.88 ± 1.02, 5.48 ± 0.34; 1.39 ± 0.21. d Verification of specificity of the anti-K-Hcy antibody. Western blotting assay was carried out by incubating the anti-KHcy antibody with unmodified OVA (ovalbumin), acetylated-OVA, succinylated-OVA, or K-Hcy-OVA. e Western blotting analysis for the detection of H3 homocystylations in samples from a variety of human fetal tissues, including brain, spinal cord, heart, liver, lung, kidney, muscle, and skin. Anti-Hcy: rabbit polyclonal anti-Hcy antibody; Anti-H3: rabbit polyclonal anti-H3 antibody. f Presence of H3 homocysteinylation in different species, including D. melanogaster , Zebra fish, Gallus gallus brain, mouse brain, and human fetal brain, demonstrated using western blotting with rabbit polyclonal anti-Hcy and anti-H3 antibodies
    Figure Legend Snippet: Histone homocysteinylation is a common modification among different tissues and species. a A typical HPLC-MS/MS spectra of a tryptic peptide ‘RGGVK Hcy RISGLIYEETR’ harboring H4K44 homocystylation, derived from human brain. The x and y axes represent m / z and relative ion intensity, respectively. A series of b- and y-type homocysteinylation fragment ions are evident which not only provide reliable sequence information, but also indicate an unambiguous +174.04600 Da shift for Hcy. b Schematic illustration of homocysteinylation sites of histone lysine residues in human normal brain samples identified using HPLC-MS/MS. The red diamond shape depicts homocysteinylation sites in core histones (H3, H4, H2a, and H2b). The number underneath each red lysine residue (K) represents the position of the particular lysine residue within each respective histone. c Verification of anti-KHcy antibody. The homocysteinylation levels of BSA (Bovine Serum Album) and KHcy modified BSA were detected with anti-KHcy antibody under the presence of 0, 2, or 5 µg/ml of Hcy modified lysine (KHcy as competitor). CBB Coomassie Brilliant Blue staining. These test repeated for 3 times and the quantitation of the western blotting showed on right. In the BSA group, the relative K-Hcy levels were 1 ± 0.05, 1.15 ± 0.10; 1.11 ± 0.78. In the BSA-Hcy group, the relative K-Hcy levels were 10.88 ± 1.02, 5.48 ± 0.34; 1.39 ± 0.21. d Verification of specificity of the anti-K-Hcy antibody. Western blotting assay was carried out by incubating the anti-KHcy antibody with unmodified OVA (ovalbumin), acetylated-OVA, succinylated-OVA, or K-Hcy-OVA. e Western blotting analysis for the detection of H3 homocystylations in samples from a variety of human fetal tissues, including brain, spinal cord, heart, liver, lung, kidney, muscle, and skin. Anti-Hcy: rabbit polyclonal anti-Hcy antibody; Anti-H3: rabbit polyclonal anti-H3 antibody. f Presence of H3 homocysteinylation in different species, including D. melanogaster , Zebra fish, Gallus gallus brain, mouse brain, and human fetal brain, demonstrated using western blotting with rabbit polyclonal anti-Hcy and anti-H3 antibodies

    Techniques Used: Modification, High Performance Liquid Chromatography, Mass Spectrometry, Derivative Assay, Sequencing, Staining, Quantitation Assay, Western Blot, Fluorescence In Situ Hybridization

    Direct in vitro histone homocysteinylation by HTL. a Schematic illustration of protein modification by HTL. b Dot-blot analysis of histone homocysteinylation by HTL. The unmodified histones H3, H4, H2a, and H2b expressed from E. coli were used. Top panel: four histones were incubated with 5 mM HTL for 2 h and histone homocysteinylation was detected using anti-Hcy antibodies. ( + : positive control, tubulin antibody diluted 1:1000 was used as the positive control; – : negative control, sodium phosphate buffer was used as the negative control); Middle panel: histone H3 was treated with 5 mM HTL for 2, 6, and 14 h, respectively, and histone homocysteinylation was detected using anti-Hcy antibodies. Bottom panel: histone H3 was treated with 0.5 mM, 1 mM, 5 mM, and 10 mM HTL respectively for 2 h and histone homocysteinylation was detected using anti-Hcy antibodies. c MALDI analysis of unmodified H3 from E. coli with (bottom) or without (top) in vitro HTL treatment. The undigested H3 display a major peak of about 15KD. Additional major peaks greater than 15KD are seen in HTL-treated H3 samples. The difference in molecular mass between the adjacent two peaks is in the proximity of 3 Hcy modifications, indicating that multiple, simultaneous KHcy modifications may exist on H3 during HTL treatment. The x and y axes represent m / z and relative ion intensity, respectively. d A typical HPLC-MS/MS spectra of a tryptic peptide ‘GVLK Hcy VFLENVIR’ derived from HTL-treated H4 with homocystylation at H4K59 site. The x and y axes represent m / z and relative ion intensity, respectively. A series of b- and y-type homocysteinylation fragment ions are evident which not only provide reliable sequence information, but also indicate an unambiguous +174.04600 Da shift for Hcy. e Illustration of histone homocysteinylation sites identified by HPLC-MS/MS analysis on unmodified core histones treated with HTL. The green diamond shape depicts homocysteinylation sites in core histones (H3, H4, H2a, and H2b). The number underneath each red lysine residue (K) represents the position of the particular lysine residue within each respective histone. Homocysteinylation sites, present both naturally in normal human brain samples (Fig. 1b ) and after in vitro HTL treatment are marked with a red dot
    Figure Legend Snippet: Direct in vitro histone homocysteinylation by HTL. a Schematic illustration of protein modification by HTL. b Dot-blot analysis of histone homocysteinylation by HTL. The unmodified histones H3, H4, H2a, and H2b expressed from E. coli were used. Top panel: four histones were incubated with 5 mM HTL for 2 h and histone homocysteinylation was detected using anti-Hcy antibodies. ( + : positive control, tubulin antibody diluted 1:1000 was used as the positive control; – : negative control, sodium phosphate buffer was used as the negative control); Middle panel: histone H3 was treated with 5 mM HTL for 2, 6, and 14 h, respectively, and histone homocysteinylation was detected using anti-Hcy antibodies. Bottom panel: histone H3 was treated with 0.5 mM, 1 mM, 5 mM, and 10 mM HTL respectively for 2 h and histone homocysteinylation was detected using anti-Hcy antibodies. c MALDI analysis of unmodified H3 from E. coli with (bottom) or without (top) in vitro HTL treatment. The undigested H3 display a major peak of about 15KD. Additional major peaks greater than 15KD are seen in HTL-treated H3 samples. The difference in molecular mass between the adjacent two peaks is in the proximity of 3 Hcy modifications, indicating that multiple, simultaneous KHcy modifications may exist on H3 during HTL treatment. The x and y axes represent m / z and relative ion intensity, respectively. d A typical HPLC-MS/MS spectra of a tryptic peptide ‘GVLK Hcy VFLENVIR’ derived from HTL-treated H4 with homocystylation at H4K59 site. The x and y axes represent m / z and relative ion intensity, respectively. A series of b- and y-type homocysteinylation fragment ions are evident which not only provide reliable sequence information, but also indicate an unambiguous +174.04600 Da shift for Hcy. e Illustration of histone homocysteinylation sites identified by HPLC-MS/MS analysis on unmodified core histones treated with HTL. The green diamond shape depicts homocysteinylation sites in core histones (H3, H4, H2a, and H2b). The number underneath each red lysine residue (K) represents the position of the particular lysine residue within each respective histone. Homocysteinylation sites, present both naturally in normal human brain samples (Fig. 1b ) and after in vitro HTL treatment are marked with a red dot

    Techniques Used: In Vitro, Modification, Dot Blot, Incubation, Positive Control, Negative Control, High Performance Liquid Chromatography, Mass Spectrometry, Derivative Assay, Sequencing

    6) Product Images from "Mammalian Protein Arginine Methyltransferase 7 (PRMT7) Specifically Targets RXR Sites in Lysine- and Arginine-rich Regions *"

    Article Title: Mammalian Protein Arginine Methyltransferase 7 (PRMT7) Specifically Targets RXR Sites in Lysine- and Arginine-rich Regions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.525345

    PRMT7-catalyzed methylation of core histones with and without buffer change detected by fluorography. 4 μg of human recombinant histones H2A, H2B, H3.3, and H4, either in the supplied buffer or exchanged into the reaction buffer (see “Experimental
    Figure Legend Snippet: PRMT7-catalyzed methylation of core histones with and without buffer change detected by fluorography. 4 μg of human recombinant histones H2A, H2B, H3.3, and H4, either in the supplied buffer or exchanged into the reaction buffer (see “Experimental

    Techniques Used: Methylation, Recombinant

    Amino acid analysis of histones methylated by PRMT7. 6 μg of recombinant human H2A, H2B, H3.3, or H4 was dialyzed against the reaction buffer and then incubated for 20 h with [ 3 H]AdoMet and PRMT7 (0.26 μ m final concentration), hydrolyzed
    Figure Legend Snippet: Amino acid analysis of histones methylated by PRMT7. 6 μg of recombinant human H2A, H2B, H3.3, or H4 was dialyzed against the reaction buffer and then incubated for 20 h with [ 3 H]AdoMet and PRMT7 (0.26 μ m final concentration), hydrolyzed

    Techniques Used: Methylation, Recombinant, Incubation, Concentration Assay

    7) Product Images from "Bub1-Mediated Adaptation of the Spindle Checkpoint"

    Article Title: Bub1-Mediated Adaptation of the Spindle Checkpoint

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1001282

    Bub1-T566A mutant has intact kinase activity or kinetochore function. (A) BUB1-T566A mutation does not alter kinase activity. Cells expressing Bub1-myc or Bub1-T566A-myc were lysed and immunoprecipitated with antibody to the myc epitope. Immunoprecipitates were analyzed by performing a kinase assay with 100 µM ATP, 0.2 µCi [gamma- 32 P]ATP in the presence or absence of human histone H2A recombinant. Coomassie Brilliant Blue staining (CBB) is shown as a loading control. (B) BUB1-T566A mutant cells do not display a chromosome missegregation phenotype. The colony color assay was performed as previously described [27] , [43] . Briefly, wild-type, bub1Δ and BUB1-T566A mutant cells containing a single SUP11 -marked chromosome fragment were plated at a density of ∼200 colonies per plate on minimal (SD) medium containing a limiting amount of adenine (6 µg/mL) and grown at 30°C. A colony consists of cells containing the chromosome fragment (white) and cells that have lost it (red), resulting in a white-and-red sectored phenotype. (C) BUB1-T566A mutant cells do not lose their endogenous chromosome. Diploid strains at MAT do not mate because of codominant suppression of haploid-specific cell differentiation pathways. Loss of either the MATa or MATalpha allele results in mating competence, where mating type is determined by the remaining allele [29] , [44] . The indicated diploids cells were mated with haploid MATa (17/14) and MAT alpha (17/17) tester strains and the mating products were selected. Two independent clones of BUB1-T566A/BUB1-T566A mutant cells are shown. (D) BUB1-T566A mutant cells do not show elevated a-like faker frequency. Loss of the MATalpha locus leads to the default mating type. MATalpha cells that lose the MAT locus will mate as a-type cells [29] , [44] . Indicated MATalpha strains were mated with the MATalpha tester strains (17/17) and mating products were selected.
    Figure Legend Snippet: Bub1-T566A mutant has intact kinase activity or kinetochore function. (A) BUB1-T566A mutation does not alter kinase activity. Cells expressing Bub1-myc or Bub1-T566A-myc were lysed and immunoprecipitated with antibody to the myc epitope. Immunoprecipitates were analyzed by performing a kinase assay with 100 µM ATP, 0.2 µCi [gamma- 32 P]ATP in the presence or absence of human histone H2A recombinant. Coomassie Brilliant Blue staining (CBB) is shown as a loading control. (B) BUB1-T566A mutant cells do not display a chromosome missegregation phenotype. The colony color assay was performed as previously described [27] , [43] . Briefly, wild-type, bub1Δ and BUB1-T566A mutant cells containing a single SUP11 -marked chromosome fragment were plated at a density of ∼200 colonies per plate on minimal (SD) medium containing a limiting amount of adenine (6 µg/mL) and grown at 30°C. A colony consists of cells containing the chromosome fragment (white) and cells that have lost it (red), resulting in a white-and-red sectored phenotype. (C) BUB1-T566A mutant cells do not lose their endogenous chromosome. Diploid strains at MAT do not mate because of codominant suppression of haploid-specific cell differentiation pathways. Loss of either the MATa or MATalpha allele results in mating competence, where mating type is determined by the remaining allele [29] , [44] . The indicated diploids cells were mated with haploid MATa (17/14) and MAT alpha (17/17) tester strains and the mating products were selected. Two independent clones of BUB1-T566A/BUB1-T566A mutant cells are shown. (D) BUB1-T566A mutant cells do not show elevated a-like faker frequency. Loss of the MATalpha locus leads to the default mating type. MATalpha cells that lose the MAT locus will mate as a-type cells [29] , [44] . Indicated MATalpha strains were mated with the MATalpha tester strains (17/17) and mating products were selected.

    Techniques Used: Mutagenesis, Activity Assay, Expressing, Immunoprecipitation, Kinase Assay, Recombinant, Staining, Cell Differentiation, Clone Assay

    8) Product Images from "Recruitment of ubiquitin-activating enzyme UBA1 to DNA by poly(ADP-ribose) promotes ATR signalling"

    Article Title: Recruitment of ubiquitin-activating enzyme UBA1 to DNA by poly(ADP-ribose) promotes ATR signalling

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201800096

    Human UBA1 binds to pADPr chains. (A) Homo sapiens UBA1, H2A, and BSA were spotted on a nitrocellulose membrane, incubated with [ 32 P]-labelled pADPr, washed, and exposed to autoradiography. (B) Purified H2A, BSA, H. sapiens UBA1 (UBA1), S. cerevisiae Uba1 (ScUba1), and H. sapiens UBA6 (UBA6) were spotted on nitrocellulose membrane and incubated with purified pADPr chains. The retention of pADPr on the membrane was revealed using an anti-pADPr antibody. (C) Increasing amounts of purified H. sapiens UBA1 (UBA1) and S. cerevisiae Uba1 (ScUba1) were spotted on nitrocellulose membrane and incubated with purified pADPr chains. The retention of pADPr on the membrane was revealed using an anti-pADPr antibody. (D) Left panel: schematic representation of UBA1 with its functional domains and the six purified overlapping fragments of UBA1. Right panel: Purified MBP-UBA1 fragments resolved by PAGE and stained by Coomassie Brilliant Blue. (E) UBA1 fragments (10 pM) were spotted on nitrocellulose membrane incubated with purified pADPr chains. Immobilised pADPr was revealed using an anti-pADPr antibody. (F) Left panel: experimental scheme. MBP UBA1 (571–800) was incubated with pADPr polymers, captured on an amylose resin, washed, and eluted with maltose. Eluted MBP UBA1 (571–800) was resolved by PAGE and revealed via anti-MBP immunoblotting. Eluted pADPr was spotted on a nitrocellulose membrane and revealed using an anti-pADPr antibody. IAD, inactive adenylation domain; FCCH, first catalytic cysteine half domain; AAD, active adenylation domain; SCCH, second catalytic cysteine half domain.
    Figure Legend Snippet: Human UBA1 binds to pADPr chains. (A) Homo sapiens UBA1, H2A, and BSA were spotted on a nitrocellulose membrane, incubated with [ 32 P]-labelled pADPr, washed, and exposed to autoradiography. (B) Purified H2A, BSA, H. sapiens UBA1 (UBA1), S. cerevisiae Uba1 (ScUba1), and H. sapiens UBA6 (UBA6) were spotted on nitrocellulose membrane and incubated with purified pADPr chains. The retention of pADPr on the membrane was revealed using an anti-pADPr antibody. (C) Increasing amounts of purified H. sapiens UBA1 (UBA1) and S. cerevisiae Uba1 (ScUba1) were spotted on nitrocellulose membrane and incubated with purified pADPr chains. The retention of pADPr on the membrane was revealed using an anti-pADPr antibody. (D) Left panel: schematic representation of UBA1 with its functional domains and the six purified overlapping fragments of UBA1. Right panel: Purified MBP-UBA1 fragments resolved by PAGE and stained by Coomassie Brilliant Blue. (E) UBA1 fragments (10 pM) were spotted on nitrocellulose membrane incubated with purified pADPr chains. Immobilised pADPr was revealed using an anti-pADPr antibody. (F) Left panel: experimental scheme. MBP UBA1 (571–800) was incubated with pADPr polymers, captured on an amylose resin, washed, and eluted with maltose. Eluted MBP UBA1 (571–800) was resolved by PAGE and revealed via anti-MBP immunoblotting. Eluted pADPr was spotted on a nitrocellulose membrane and revealed using an anti-pADPr antibody. IAD, inactive adenylation domain; FCCH, first catalytic cysteine half domain; AAD, active adenylation domain; SCCH, second catalytic cysteine half domain.

    Techniques Used: Incubation, Autoradiography, Purification, Functional Assay, Polyacrylamide Gel Electrophoresis, Staining

    9) Product Images from "Structure Change from β-Strand and Turn to α-Helix in Histone H2A-H2B Induced by DNA Damage Response"

    Article Title: Structure Change from β-Strand and Turn to α-Helix in Histone H2A-H2B Induced by DNA Damage Response

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2016.06.002

    ( a ) Temperature dependence of the CD intensity at 222 nm of H2A-H2B extracted from core histones in unirradiated (Unirrad., open circle ) and x-irradiated (Irrad., solid circle ) cells. Inset: magnification of the unirradiated sample between 320
    Figure Legend Snippet: ( a ) Temperature dependence of the CD intensity at 222 nm of H2A-H2B extracted from core histones in unirradiated (Unirrad., open circle ) and x-irradiated (Irrad., solid circle ) cells. Inset: magnification of the unirradiated sample between 320

    Techniques Used: Irradiation

    Thermodynamic parameters of H2A-H2B
    Figure Legend Snippet: Thermodynamic parameters of H2A-H2B

    Techniques Used:

    ( a–c ) CD spectra of H2A-H2B extracted from core histones in unirradiated (Unirrad., black ) and x-irradiated (Irrad., red ) cells, measured at ( a ) 294, ( b ) 310, and ( c ) 330 K. In ( a ), a CD spectrum of H2A-H2B irradiated with 40 Gy x-rays
    Figure Legend Snippet: ( a–c ) CD spectra of H2A-H2B extracted from core histones in unirradiated (Unirrad., black ) and x-irradiated (Irrad., red ) cells, measured at ( a ) 294, ( b ) 310, and ( c ) 330 K. In ( a ), a CD spectrum of H2A-H2B irradiated with 40 Gy x-rays

    Techniques Used: Irradiation

    ( a–d ) Temperature dependence of the contents of ( a ) α -helix, ( b ) β -strand, ( c ) turn, and ( d ) other structures of H2A-H2B extracted from core histones in unirradiated (Unirrad., open circle ) and x-irradiated (Irrad., solid
    Figure Legend Snippet: ( a–d ) Temperature dependence of the contents of ( a ) α -helix, ( b ) β -strand, ( c ) turn, and ( d ) other structures of H2A-H2B extracted from core histones in unirradiated (Unirrad., open circle ) and x-irradiated (Irrad., solid

    Techniques Used: Irradiation

    ( a ) SDS-PAGE analysis of H2A-H2B extracted from core histones in unirradiated cells and 40 Gy irradiated cells after 30 min incubation. The lane labeled M refers to the standard molecular weight marker (XL-Ladder (low range); APRO Life
    Figure Legend Snippet: ( a ) SDS-PAGE analysis of H2A-H2B extracted from core histones in unirradiated cells and 40 Gy irradiated cells after 30 min incubation. The lane labeled M refers to the standard molecular weight marker (XL-Ladder (low range); APRO Life

    Techniques Used: SDS Page, Irradiation, Incubation, Labeling, Molecular Weight, Marker

    10) Product Images from "Differential Microbicidal Effects of Human Histone Proteins H2A and H2B on Leishmania Promastigotes and Amastigotes ▿"

    Article Title: Differential Microbicidal Effects of Human Histone Proteins H2A and H2B on Leishmania Promastigotes and Amastigotes ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00658-10

    Surface and internal binding of histone proteins on Leishmania parasites. L. amazonensis promastigotes (A) or axenic amastigotes (B) were treated with the indicated concentration of histone H2A for 30 min. After washing, surface-bound H2A was stained
    Figure Legend Snippet: Surface and internal binding of histone proteins on Leishmania parasites. L. amazonensis promastigotes (A) or axenic amastigotes (B) were treated with the indicated concentration of histone H2A for 30 min. After washing, surface-bound H2A was stained

    Techniques Used: Binding Assay, Concentration Assay, Staining

    Morphological and ultrastructural changes of histone-treated parasites in scanning electron microscopy (SEM). L. amazonensis promastigotes were treated with histone H2A or H2B (100 μg/ml) for 30 min, and then samples were immediately fixed for
    Figure Legend Snippet: Morphological and ultrastructural changes of histone-treated parasites in scanning electron microscopy (SEM). L. amazonensis promastigotes were treated with histone H2A or H2B (100 μg/ml) for 30 min, and then samples were immediately fixed for

    Techniques Used: Electron Microscopy

    Direct killing of Leishmania promastigotes, but not amastigotes, by histone proteins. Promastigotes of L. amazonensis (A and B), L. major (C), and L. braziliensis (D) were treated with 20 to 200 μg/of of histone H2A, H2B, or H1 0 for 30 min and
    Figure Legend Snippet: Direct killing of Leishmania promastigotes, but not amastigotes, by histone proteins. Promastigotes of L. amazonensis (A and B), L. major (C), and L. braziliensis (D) were treated with 20 to 200 μg/of of histone H2A, H2B, or H1 0 for 30 min and

    Techniques Used:

    Suppressive effects of histone H2A and H2B on parasite proliferation. L. amazonensis promastigotes (2.5 × 10 6 in 50 μl PBS) were treated with 20 or 100 μg/ml of histone H2A (A) or H2B (B) at 23°C for indicated time periods,
    Figure Legend Snippet: Suppressive effects of histone H2A and H2B on parasite proliferation. L. amazonensis promastigotes (2.5 × 10 6 in 50 μl PBS) were treated with 20 or 100 μg/ml of histone H2A (A) or H2B (B) at 23°C for indicated time periods,

    Techniques Used:

    Analysis of the death rates of histone-treated parasites by flow cytometry. (A) Metacyclic promastigotes of L. major and L. amazonensis were purified by using Ficoll gradient centrifugation and treated with an indicated concentration of histone H2A. Axenic
    Figure Legend Snippet: Analysis of the death rates of histone-treated parasites by flow cytometry. (A) Metacyclic promastigotes of L. major and L. amazonensis were purified by using Ficoll gradient centrifugation and treated with an indicated concentration of histone H2A. Axenic

    Techniques Used: Flow Cytometry, Cytometry, Purification, Gradient Centrifugation, Concentration Assay

    Mφ infection with histone-treated L. amazonensis parasites. (A and B) L. amazonensis promastigotes were treated with an indicated concentration of H2A or H2B for 30 min and used to infect Mφs (at a 5:1 parasite-to-cell ratio). The number
    Figure Legend Snippet: Mφ infection with histone-treated L. amazonensis parasites. (A and B) L. amazonensis promastigotes were treated with an indicated concentration of H2A or H2B for 30 min and used to infect Mφs (at a 5:1 parasite-to-cell ratio). The number

    Techniques Used: Infection, Concentration Assay

    Effects of LPG and gp63 on histone-mediated promastigote killing. (A) Wild-type (WT), lpg1 − , and lpg1 − /+ LPG1 promastigotes of L. major were treated with the indicated concentrations (μg/ml) of histone H2A or H2B and then
    Figure Legend Snippet: Effects of LPG and gp63 on histone-mediated promastigote killing. (A) Wild-type (WT), lpg1 − , and lpg1 − /+ LPG1 promastigotes of L. major were treated with the indicated concentrations (μg/ml) of histone H2A or H2B and then

    Techniques Used:

    11) Product Images from "Breaching peripheral tolerance promotes the production of HIV-1–neutralizing antibodies"

    Article Title: Breaching peripheral tolerance promotes the production of HIV-1–neutralizing antibodies

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20161190

    Histone H2A-reactive IgM monoclonal antibodies, isolated from B6.Sle123 mice, neutralize tier 2 strains of HIV-1. Hybridomas were generated from splenocytes isolated from B6.Sle123 mice that displayed serum tier 2 HIV-1 neutralization ( n = 2). (A) Purified monoclonal IgM and IgG antibodies ( n = 8) were tested for HIV-1 neutralization against 4 strains of HIV-1 and reported as IC 50 (the concentration of antibody required for 50% neutralization). (B) Specificities of the monoclonal antibodies were tested using ELISA against the histone H2A, histone H2B, and chromatin nuclear antigens in addition to HIV-1 gp140 (YU2) Env and the CD4bs (RSC3) epitope, as well as a CD4bs-negative control (ΔRSC3). The two neutralizing IgM mAbs (P4E4 and O4C5) are shown with red symbols, and lines and non-neutralizing mAbs shown with solid black symbols and lines for IgM and gray symbols and dashed lines for IgG. IgM and IgG control antibodies were used to determine the background of the assay and for which ODs above this line were considered positive. Data are representative from three independent experiments.
    Figure Legend Snippet: Histone H2A-reactive IgM monoclonal antibodies, isolated from B6.Sle123 mice, neutralize tier 2 strains of HIV-1. Hybridomas were generated from splenocytes isolated from B6.Sle123 mice that displayed serum tier 2 HIV-1 neutralization ( n = 2). (A) Purified monoclonal IgM and IgG antibodies ( n = 8) were tested for HIV-1 neutralization against 4 strains of HIV-1 and reported as IC 50 (the concentration of antibody required for 50% neutralization). (B) Specificities of the monoclonal antibodies were tested using ELISA against the histone H2A, histone H2B, and chromatin nuclear antigens in addition to HIV-1 gp140 (YU2) Env and the CD4bs (RSC3) epitope, as well as a CD4bs-negative control (ΔRSC3). The two neutralizing IgM mAbs (P4E4 and O4C5) are shown with red symbols, and lines and non-neutralizing mAbs shown with solid black symbols and lines for IgM and gray symbols and dashed lines for IgG. IgM and IgG control antibodies were used to determine the background of the assay and for which ODs above this line were considered positive. Data are representative from three independent experiments.

    Techniques Used: Isolation, Mouse Assay, Generated, Neutralization, Purification, Concentration Assay, Enzyme-linked Immunosorbent Assay, Negative Control

    Elevated IgM anti-histone H2A titers correlate with tier 2 HIV-1 neutralization by pristane treated wild-type C57BL/6 mice. Total serum concentrations of (A) IgM, (B) IgG, and (C) relative titers of serum IgM anti-H2A are shown for naive (open circles) B6 mice or B6 mice treated 30 d with pristane only (gray circles) and subsequently immunized 2X or 3X (black circles) with alum alone or Env + alum. Serum from individual B6 mice (regardless of treatment with pristane alone, alum alone, or Env + alum) were separated based on neutralization of ≥1 tier 2 HIV-1 strains and measured for (D) IgM anti-H2A or (E) IgM anti-H2B relative titers. Mice neutralizing only tier 1 strains were included in the tier 2 nonneutralizer group (mostly 3X). Each symbol represents measurements for one mouse, and all data are plotted as the arithmetic mean ± SEM. All P-values were calculated using Student’s t test assuming unequal variances. *, P
    Figure Legend Snippet: Elevated IgM anti-histone H2A titers correlate with tier 2 HIV-1 neutralization by pristane treated wild-type C57BL/6 mice. Total serum concentrations of (A) IgM, (B) IgG, and (C) relative titers of serum IgM anti-H2A are shown for naive (open circles) B6 mice or B6 mice treated 30 d with pristane only (gray circles) and subsequently immunized 2X or 3X (black circles) with alum alone or Env + alum. Serum from individual B6 mice (regardless of treatment with pristane alone, alum alone, or Env + alum) were separated based on neutralization of ≥1 tier 2 HIV-1 strains and measured for (D) IgM anti-H2A or (E) IgM anti-H2B relative titers. Mice neutralizing only tier 1 strains were included in the tier 2 nonneutralizer group (mostly 3X). Each symbol represents measurements for one mouse, and all data are plotted as the arithmetic mean ± SEM. All P-values were calculated using Student’s t test assuming unequal variances. *, P

    Techniques Used: Neutralization, Mouse Assay

    B6.Sle123 HIV-1 neutralizers harbor elevated levels of IgM anti-histone H2A. (A) Sera from B6 (open), B6.Sle123 nonneutralizers (gray), and neutralizers (black) were interrogated with an autoantigen array and results for the IgM reactive with the indicated anti-DNA antigens (top left), RNA-binding proteins (bottom left), and anti-histone antigens (top right), as well as IgG anti-histone antigens (bottom right) are shown. Relative serum titers of (B) IgM anti-H2A (left) and anti-H2B (right) measured by ELISA in B6.Sle123 neutralizers and nonneutralizers. Each symbol represents measurements for one mouse, and all data are plotted as the arithmetic mean ± SEM. P-values were calculated using the Mann-Whitney nonparametric test; *, P
    Figure Legend Snippet: B6.Sle123 HIV-1 neutralizers harbor elevated levels of IgM anti-histone H2A. (A) Sera from B6 (open), B6.Sle123 nonneutralizers (gray), and neutralizers (black) were interrogated with an autoantigen array and results for the IgM reactive with the indicated anti-DNA antigens (top left), RNA-binding proteins (bottom left), and anti-histone antigens (top right), as well as IgG anti-histone antigens (bottom right) are shown. Relative serum titers of (B) IgM anti-H2A (left) and anti-H2B (right) measured by ELISA in B6.Sle123 neutralizers and nonneutralizers. Each symbol represents measurements for one mouse, and all data are plotted as the arithmetic mean ± SEM. P-values were calculated using the Mann-Whitney nonparametric test; *, P

    Techniques Used: RNA Binding Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    12) Product Images from "PRMT5 interacts with the BCL6 oncoprotein and is required for germinal center formation and lymphoma cell survival"

    Article Title: PRMT5 interacts with the BCL6 oncoprotein and is required for germinal center formation and lymphoma cell survival

    Journal: Blood

    doi: 10.1182/blood-2018-02-831438

    PRMT5 directly dimethylates arginines of BCL6 in lymphoma. (A) IP for BCL6 and symmetric arginine dimethylation (SYM10) in lymphoma cell lines OCI-LY1 and Raji reveals symmetric arginine dimethylation of BCL6. (B) Knockdown of PRMT5 with specific siRNA decreases BCL6 symmetric arginine methylation. Raji cells were transfected with PRMT5 or control siRNAs followed by IP with symmetric arginine dimethylation (SYM10) antibody and immunoblotting with BCL6 antibody. Also shown are western blots with indicated antibodies from the same cells. (C) In vitro methyltransferase assay with recombinant PRMT5, MEP50, and BCL6 or H2A proteins. The proteins were blotted with the indicated antibodies. In addition, the reaction mixture was immunoprecipitated with BCL6 antibody and blotted with antibodies for symmetric arginine dimethylation (SYM10) and BCL6. (D) In vitro thymidine incorporation methyltransferase assay with recombinant PRMT5 and BCL6 or H2A proteins.
    Figure Legend Snippet: PRMT5 directly dimethylates arginines of BCL6 in lymphoma. (A) IP for BCL6 and symmetric arginine dimethylation (SYM10) in lymphoma cell lines OCI-LY1 and Raji reveals symmetric arginine dimethylation of BCL6. (B) Knockdown of PRMT5 with specific siRNA decreases BCL6 symmetric arginine methylation. Raji cells were transfected with PRMT5 or control siRNAs followed by IP with symmetric arginine dimethylation (SYM10) antibody and immunoblotting with BCL6 antibody. Also shown are western blots with indicated antibodies from the same cells. (C) In vitro methyltransferase assay with recombinant PRMT5, MEP50, and BCL6 or H2A proteins. The proteins were blotted with the indicated antibodies. In addition, the reaction mixture was immunoprecipitated with BCL6 antibody and blotted with antibodies for symmetric arginine dimethylation (SYM10) and BCL6. (D) In vitro thymidine incorporation methyltransferase assay with recombinant PRMT5 and BCL6 or H2A proteins.

    Techniques Used: Methylation, Transfection, Western Blot, In Vitro, Recombinant, Immunoprecipitation

    13) Product Images from "ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair"

    Article Title: ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201506099

    ZRF1 regulates XPC ubiquitylation. (A) ZRF1 facilitates XPC ubiquitylation after UV irradiation. Whole-cell extracts of control and ZRF1 knockdown HEK293T cells from the stated time points were subjected to Western blotting and probed with the indicated antibodies. (B) Role of RING1B and ZRF1 in XPC ubiquitylation. Control cells and RING1B and ZRF1 knockdown HEK293T cells expressing HA Ubiquitin were irradiated with UV light. After immunoprecipitation with HA-specific antibody, the precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (C) Control cells and RING1B and ZRF1 knockdown HEK293T cells expressing HA XPC and HIS Ubiquitin were irradiated with UV light. After immunoprecipitation with HA-specific antibody, the precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (D) Control cells and RING1B and ZRF1 knockdown HEK293T cells expressing HISUbiquitin were irradiated with UV and harvested 1 h after UV exposure. Ubiquitylated proteins were purified by NiNTA agarose under denaturing conditions, and Western blots of the purified material were incubated with the indicated antibodies. (E) The UV–RING1B complex and ZRF1 cooperate during NER. DNA lesions (yellow star) are recognized by the UV-RING1B complex (DDB1–DDB2–CUL4B–RING1B), which catalyzes ubiquitylation of histone H2A (gray sphere). ZRF1 is recruited to the lesion site by XPC and tethers to the H2A-ubiquitin mark. ZRF1 causes the assembly of the UV–DDB–CUL4A complex, which subsequently catalyzes ubiquitylation of XPC.
    Figure Legend Snippet: ZRF1 regulates XPC ubiquitylation. (A) ZRF1 facilitates XPC ubiquitylation after UV irradiation. Whole-cell extracts of control and ZRF1 knockdown HEK293T cells from the stated time points were subjected to Western blotting and probed with the indicated antibodies. (B) Role of RING1B and ZRF1 in XPC ubiquitylation. Control cells and RING1B and ZRF1 knockdown HEK293T cells expressing HA Ubiquitin were irradiated with UV light. After immunoprecipitation with HA-specific antibody, the precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (C) Control cells and RING1B and ZRF1 knockdown HEK293T cells expressing HA XPC and HIS Ubiquitin were irradiated with UV light. After immunoprecipitation with HA-specific antibody, the precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (D) Control cells and RING1B and ZRF1 knockdown HEK293T cells expressing HISUbiquitin were irradiated with UV and harvested 1 h after UV exposure. Ubiquitylated proteins were purified by NiNTA agarose under denaturing conditions, and Western blots of the purified material were incubated with the indicated antibodies. (E) The UV–RING1B complex and ZRF1 cooperate during NER. DNA lesions (yellow star) are recognized by the UV-RING1B complex (DDB1–DDB2–CUL4B–RING1B), which catalyzes ubiquitylation of histone H2A (gray sphere). ZRF1 is recruited to the lesion site by XPC and tethers to the H2A-ubiquitin mark. ZRF1 causes the assembly of the UV–DDB–CUL4A complex, which subsequently catalyzes ubiquitylation of XPC.

    Techniques Used: Irradiation, Western Blot, Expressing, Immunoprecipitation, Incubation, Purification

    RING1B and DDB2 cooperate in H2A ubiquitylation. (A) RING1B interacts with DDB2. Control cells and cells expressing FLAG RING1B were irradiated with UV light. After immunoprecipitation with FLAG-M2-Agarose the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (B) Endogenous immunoprecipitations with RING1B antibodies after UV irradiation. Western blots of the precipitated material were incubated with the indicated antibodies. IgG lanes show unspecific staining of the IgG heavy chains. (C) DDB2 associates with RING1B. Control cells and cells expressing FLAG DDB2 were irradiated with UV light. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (D) Epistatic relationship of RING1B and DDB2 in response to UV irradiation. Relative colony formation potential of control or RING1B knockdown cell lines treated with siRNA was analyzed at different UV dosages. Control cells were transfected with either control siRNA (control) or DDB2 siRNA ( DDB2 ). RING1B knockdown cell lines were transfected with either control siRNA ( RING1B ) or DDB2 siRNA ( RING1B + DDB2 ). Gene knockdown was confirmed by Western blots (not depicted). Values are given as mean ± SEM ( n = 6). (E) Knockdown of RING1B does not impair DDB2 recruitment. Chromatin association assays of control and RING1B knockdown HEK293T cells after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative DDB2 and BMI-1 abundance was calculated. Values are given as mean ± SEM ( n = 3). (F) Knockdown of DDB2 shows reduced H2A-ubiquitylation but unaltered BMI-1 recruitment. Chromatin association assays of UV-irradiated HEK293T cells treated with siRNAs (control, DDB2 ). De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A-ubiquitylation and RING1B abundance was calculated. Values are given as mean ± SEM ( n = 4).
    Figure Legend Snippet: RING1B and DDB2 cooperate in H2A ubiquitylation. (A) RING1B interacts with DDB2. Control cells and cells expressing FLAG RING1B were irradiated with UV light. After immunoprecipitation with FLAG-M2-Agarose the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (B) Endogenous immunoprecipitations with RING1B antibodies after UV irradiation. Western blots of the precipitated material were incubated with the indicated antibodies. IgG lanes show unspecific staining of the IgG heavy chains. (C) DDB2 associates with RING1B. Control cells and cells expressing FLAG DDB2 were irradiated with UV light. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (D) Epistatic relationship of RING1B and DDB2 in response to UV irradiation. Relative colony formation potential of control or RING1B knockdown cell lines treated with siRNA was analyzed at different UV dosages. Control cells were transfected with either control siRNA (control) or DDB2 siRNA ( DDB2 ). RING1B knockdown cell lines were transfected with either control siRNA ( RING1B ) or DDB2 siRNA ( RING1B + DDB2 ). Gene knockdown was confirmed by Western blots (not depicted). Values are given as mean ± SEM ( n = 6). (E) Knockdown of RING1B does not impair DDB2 recruitment. Chromatin association assays of control and RING1B knockdown HEK293T cells after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative DDB2 and BMI-1 abundance was calculated. Values are given as mean ± SEM ( n = 3). (F) Knockdown of DDB2 shows reduced H2A-ubiquitylation but unaltered BMI-1 recruitment. Chromatin association assays of UV-irradiated HEK293T cells treated with siRNAs (control, DDB2 ). De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A-ubiquitylation and RING1B abundance was calculated. Values are given as mean ± SEM ( n = 4).

    Techniques Used: Expressing, Irradiation, Immunoprecipitation, Purification, Western Blot, Incubation, Staining, Transfection

    ZRF1 facilitates the assembly of the UV – DDB – CUL4A E3 ligase complex. (A) ZRF1 displaces RING1B from chromatin during NER. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A ubiquitin and RING1B abundance was calculated. Values are given as mean ± SEM ( n = 3). (B) ZRF1 regulates chromatin association of CUL4A and CUL4B. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative CUL4B and CUL4A abundance was calculated. Values are given as mean ± SEM ( n = 3). (C) ZRF1 regulates CUL4A association with H2AX containing nucleosomes. Control cells and ZRF1 knockdown cells expressing FLAG H2AX were irradiated with UV. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (D) Knockdown of ZRF1 modulates CUL4A association with DDB2. Control cells and ZRF1 knockdown cells expressing FLAG DDB2 were irradiated with UV light. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (E) Assembly of the UV–DDB–CUL4A E3 ligase is facilitated by ZRF1. Control cells and ZRF1 knockdown HEK293T cells expressing HA RBX1 were irradiated with UV light. After immunoprecipitation with HA-specific antibodies the precipitated material was subjected to Western blotting, and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (F) ZRF1 competes with CUL4B and RING1B for DDB2 binding in vitro. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4B, and RING1B and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 over the other components (relative molarity ZRF1: DDB1–CUL4B–RING1B; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (G) ZRF1 does not compete with CUL4A and RBX1 for binding to DDB1–DDB2. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4A and RBX1 and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 (relative molarity ZRF1: DDB1–CUL4A–RBX1; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (H) ZRF1 mediates the formation of the UV-DDB-CUL4A complex in vitro. GFP and GFP-DDB2 were coupled to beads and incubated with CUL4B, DDB1 and RING1B. After washing, GFP and GFP-DDB2 (UV–RING1B complex) beads were incubated with an estimated fivefold excess of purified CUL4A and RBX1 (lanes 1–3) over the retained UV–RING1B complex. Simultaneously, ZRF1 (lanes 1 and 3) or GST (lane 2) was added to the incubations in equimolar amounts. The precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%.
    Figure Legend Snippet: ZRF1 facilitates the assembly of the UV – DDB – CUL4A E3 ligase complex. (A) ZRF1 displaces RING1B from chromatin during NER. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A ubiquitin and RING1B abundance was calculated. Values are given as mean ± SEM ( n = 3). (B) ZRF1 regulates chromatin association of CUL4A and CUL4B. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative CUL4B and CUL4A abundance was calculated. Values are given as mean ± SEM ( n = 3). (C) ZRF1 regulates CUL4A association with H2AX containing nucleosomes. Control cells and ZRF1 knockdown cells expressing FLAG H2AX were irradiated with UV. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (D) Knockdown of ZRF1 modulates CUL4A association with DDB2. Control cells and ZRF1 knockdown cells expressing FLAG DDB2 were irradiated with UV light. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (E) Assembly of the UV–DDB–CUL4A E3 ligase is facilitated by ZRF1. Control cells and ZRF1 knockdown HEK293T cells expressing HA RBX1 were irradiated with UV light. After immunoprecipitation with HA-specific antibodies the precipitated material was subjected to Western blotting, and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (F) ZRF1 competes with CUL4B and RING1B for DDB2 binding in vitro. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4B, and RING1B and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 over the other components (relative molarity ZRF1: DDB1–CUL4B–RING1B; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (G) ZRF1 does not compete with CUL4A and RBX1 for binding to DDB1–DDB2. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4A and RBX1 and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 (relative molarity ZRF1: DDB1–CUL4A–RBX1; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (H) ZRF1 mediates the formation of the UV-DDB-CUL4A complex in vitro. GFP and GFP-DDB2 were coupled to beads and incubated with CUL4B, DDB1 and RING1B. After washing, GFP and GFP-DDB2 (UV–RING1B complex) beads were incubated with an estimated fivefold excess of purified CUL4A and RBX1 (lanes 1–3) over the retained UV–RING1B complex. Simultaneously, ZRF1 (lanes 1 and 3) or GST (lane 2) was added to the incubations in equimolar amounts. The precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%.

    Techniques Used: Irradiation, Western Blot, Expressing, Immunoprecipitation, Purification, Incubation, Binding Assay, In Vitro

    ZRF1 interacts with XPC during UV-mediated DNA repair. (A) ZRF1 specifically binds to XPC. Control and FLAG ZRF1-expressing cells were irradiated with UV light. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 4%. (B) Endogenous immunopreciptiations with ZRF1 antibodies. Precipitates were subjected to Western blotting, and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (C) ZRF1 localization to DNA damage sites is dependent on XPC. Control fibroblasts and XPC patient fibroblasts expressing both mCherry-ZRF1 and DDB2-GFP were UV irradiated (100 J/m 2 ) through a micropore membrane. Thirty minutes after irradiation, cells were preextracted and fixed. DNA damage sites were visualized by DDB2-GFP. (D) ZRF1 enriches at chromatin after UV irradiation in a XPC-dependent manner. Chromatin association assays with control fibroblasts (GM16248) and XPC patient fibroblasts (GM15983) after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A-ubiquitin and ZRF1 abundance was calculated. Values are given as mean ± SEM ( n = 3). (E) H2A ubiquitylation is not altered in XPA patient fibroblasts. Chromatin association assays with control fibroblasts (GM15876) and XPA fibroblasts (GM04312) after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. Relative intensities of H2A-ubiquitin/H2A, ZRF1 and RING1B abundance were measured. Values are given as mean ± SEM ( n = 3). (F) Epistasis analysis of ZRF1 and XPC. The relative colony formation potential of control or ZRF1 knockdown cell lines treated with control (Control; ZRF1 ) or XPC siRNA ( XPC ; ZRF1+XPC ) was analyzed at different UV doses. Gene knockdown was confirmed by Western blots (not depicted). Values are given as mean ± SEM ( n = 3).
    Figure Legend Snippet: ZRF1 interacts with XPC during UV-mediated DNA repair. (A) ZRF1 specifically binds to XPC. Control and FLAG ZRF1-expressing cells were irradiated with UV light. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 4%. (B) Endogenous immunopreciptiations with ZRF1 antibodies. Precipitates were subjected to Western blotting, and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (C) ZRF1 localization to DNA damage sites is dependent on XPC. Control fibroblasts and XPC patient fibroblasts expressing both mCherry-ZRF1 and DDB2-GFP were UV irradiated (100 J/m 2 ) through a micropore membrane. Thirty minutes after irradiation, cells were preextracted and fixed. DNA damage sites were visualized by DDB2-GFP. (D) ZRF1 enriches at chromatin after UV irradiation in a XPC-dependent manner. Chromatin association assays with control fibroblasts (GM16248) and XPC patient fibroblasts (GM15983) after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A-ubiquitin and ZRF1 abundance was calculated. Values are given as mean ± SEM ( n = 3). (E) H2A ubiquitylation is not altered in XPA patient fibroblasts. Chromatin association assays with control fibroblasts (GM15876) and XPA fibroblasts (GM04312) after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. Relative intensities of H2A-ubiquitin/H2A, ZRF1 and RING1B abundance were measured. Values are given as mean ± SEM ( n = 3). (F) Epistasis analysis of ZRF1 and XPC. The relative colony formation potential of control or ZRF1 knockdown cell lines treated with control (Control; ZRF1 ) or XPC siRNA ( XPC ; ZRF1+XPC ) was analyzed at different UV doses. Gene knockdown was confirmed by Western blots (not depicted). Values are given as mean ± SEM ( n = 3).

    Techniques Used: Expressing, Irradiation, Immunoprecipitation, Purification, Western Blot, Incubation

    H2A ubiquitylation after UV irradiation is performed by the UV–RING1B complex. (A) Protein interaction partners of RING1B and DDB2. Mass spectrometry analysis after sequential immunoprecipitations with FLAG and RING1B antibodies revealed DDB1 and CUL4B as main interaction partners of DDB2 and RING1B. A comprehensive list of the identified unique peptides after RING1B and control immunoprecipitations (with or without UV irradiation) is provided in Table S5 . (B) Assembly of the UV–RING1B complex. Plasmids expressing FLAG DDB1, FLAG DDB2, and FLAG RING1B were cotransfected in combination with either control plasmid or a plasmid encoding FLAG-STREP CUL4B. After immunoprecipitation with STREP-Tactin beads, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (C) Visualization of the UV–RING1B complex. Purified UV–RING1B complex was subjected to SDS gel electrophoresis and colloidal Coomassie staining. Mass spectrometry analysis revealed the presence of all four subunits (bold). A comprehensive list of unique peptides is provided in Table S6 . (D) The UV–RING1B complex catalyzes ubiquitylation of H2A in vitro. Ubiquitylation assays were performed with recombinant H2A, E1 (UBA1), E2 (UBCH5), and either GST (control) or the UV–RING1B complex. Reactions were performed at 37°C, and samples were taken at the indicated time points. Material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. (E) The UV–RING1B complex catalyzes monoubiquitylation of nucleosomal H2A. Ubiquitylation assays were performed with recombinant nucleosomes, E1 (UBA1), E2 (UBCH5), and either GST (control) or UV-RING1B complex. Reactions lacking E1 (−E1) were performed as additional controls. The ubiquitylation assays were performed at 37°C for 5 h, and samples or pure substrate (Substrate) were subjected to Western blotting and probed with H2A antibodies.
    Figure Legend Snippet: H2A ubiquitylation after UV irradiation is performed by the UV–RING1B complex. (A) Protein interaction partners of RING1B and DDB2. Mass spectrometry analysis after sequential immunoprecipitations with FLAG and RING1B antibodies revealed DDB1 and CUL4B as main interaction partners of DDB2 and RING1B. A comprehensive list of the identified unique peptides after RING1B and control immunoprecipitations (with or without UV irradiation) is provided in Table S5 . (B) Assembly of the UV–RING1B complex. Plasmids expressing FLAG DDB1, FLAG DDB2, and FLAG RING1B were cotransfected in combination with either control plasmid or a plasmid encoding FLAG-STREP CUL4B. After immunoprecipitation with STREP-Tactin beads, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (C) Visualization of the UV–RING1B complex. Purified UV–RING1B complex was subjected to SDS gel electrophoresis and colloidal Coomassie staining. Mass spectrometry analysis revealed the presence of all four subunits (bold). A comprehensive list of unique peptides is provided in Table S6 . (D) The UV–RING1B complex catalyzes ubiquitylation of H2A in vitro. Ubiquitylation assays were performed with recombinant H2A, E1 (UBA1), E2 (UBCH5), and either GST (control) or the UV–RING1B complex. Reactions were performed at 37°C, and samples were taken at the indicated time points. Material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. (E) The UV–RING1B complex catalyzes monoubiquitylation of nucleosomal H2A. Ubiquitylation assays were performed with recombinant nucleosomes, E1 (UBA1), E2 (UBCH5), and either GST (control) or UV-RING1B complex. Reactions lacking E1 (−E1) were performed as additional controls. The ubiquitylation assays were performed at 37°C for 5 h, and samples or pure substrate (Substrate) were subjected to Western blotting and probed with H2A antibodies.

    Techniques Used: Irradiation, Mass Spectrometry, Expressing, Plasmid Preparation, Immunoprecipitation, Purification, Western Blot, Incubation, SDS-Gel, Electrophoresis, Staining, In Vitro, Recombinant

    Function of ZRF1 in UV-mediated DNA repair. (A) ZRF1 is tethered to chromatin in a RING1B-dependent manner. Chromatin association assays of control and RING1B knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative ZRF1 abundance was calculated. Values are given as mean ± SEM ( n = 3). (B) The ubiquitin-binding domain (UBD) is important for tethering ZRF1 to chromatin after UV irradiation. HEK293T cells expressing FLAG ZRF1 and FLAG ZRF1-ΔUBD were irradiated with UV light, and chromatin was isolated at the indicated time points. De-cross-linked material was subjected to Western blotting and blots were incubated with FLAG-antibody. The relative FLAG ZRF1 abundance was calculated. Values are given as mean ± SEM ( n = 4). (C and D) ZRF1 localizes to DNA damage sites after UV irradiation. MRC5 fibroblasts expressing mCherry-ZRF1 were UV irradiated (100 J/m 2 ) through a micropore membrane (+ UV) 24 h after transfection. 30 min after irradiation, cells were preextracted and fixed. DNA damage sites were visualized by staining with XPC (C) or XPA (D) antibody. The colocalization of ZRF1 with XPC amounts to 88% ± 1%. The colocalization of ZRF1 with XPA amounts to 73% ± −3%. Nonirradiated control and quantification of the ZRF1 localization at the damage sites are represented in Fig. S4 A . Bar, 10 µm. (E) Inhibition of RING1B affects recruitment of ZRF1 to DNA damage sites. MRC5 fibroblasts expressing mCherry-ZRF1 were treated with PRT4165 or DMSO. Cells were UV-irradiated (100 J/m 2 ) through a micropore membrane. 30 min after irradiation cells were preextracted and fixed. DNA damage sites were visualized by XPC antibody staining. ZRF1 localization to DNA lesions after treatment with DMSO or PRT4165 was quantified (Fig. S4 B). Bar, 10 µm. (F) Depletion of CUL4B impacts H2A ubiquitylation and ZRF1 recruitment. Chromatin association assays of UV irradiated HEK293T cells treated with siRNAs (control, CUL4B ). De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A-ubiquitin and ZRF1 abundance was calculated. Values are given as mean ± SEM ( n = 3). (G) Tethering of ZRF1 to chromatin depends on DDB2 during NER. Chromatin association assays in control fibroblasts (GM15876) and XPE (DDB2) fibroblasts (GM01389) after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative RING1B and ZRF1 abundance was calculated. Values are given as mean ± SEM ( n = 3).
    Figure Legend Snippet: Function of ZRF1 in UV-mediated DNA repair. (A) ZRF1 is tethered to chromatin in a RING1B-dependent manner. Chromatin association assays of control and RING1B knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative ZRF1 abundance was calculated. Values are given as mean ± SEM ( n = 3). (B) The ubiquitin-binding domain (UBD) is important for tethering ZRF1 to chromatin after UV irradiation. HEK293T cells expressing FLAG ZRF1 and FLAG ZRF1-ΔUBD were irradiated with UV light, and chromatin was isolated at the indicated time points. De-cross-linked material was subjected to Western blotting and blots were incubated with FLAG-antibody. The relative FLAG ZRF1 abundance was calculated. Values are given as mean ± SEM ( n = 4). (C and D) ZRF1 localizes to DNA damage sites after UV irradiation. MRC5 fibroblasts expressing mCherry-ZRF1 were UV irradiated (100 J/m 2 ) through a micropore membrane (+ UV) 24 h after transfection. 30 min after irradiation, cells were preextracted and fixed. DNA damage sites were visualized by staining with XPC (C) or XPA (D) antibody. The colocalization of ZRF1 with XPC amounts to 88% ± 1%. The colocalization of ZRF1 with XPA amounts to 73% ± −3%. Nonirradiated control and quantification of the ZRF1 localization at the damage sites are represented in Fig. S4 A . Bar, 10 µm. (E) Inhibition of RING1B affects recruitment of ZRF1 to DNA damage sites. MRC5 fibroblasts expressing mCherry-ZRF1 were treated with PRT4165 or DMSO. Cells were UV-irradiated (100 J/m 2 ) through a micropore membrane. 30 min after irradiation cells were preextracted and fixed. DNA damage sites were visualized by XPC antibody staining. ZRF1 localization to DNA lesions after treatment with DMSO or PRT4165 was quantified (Fig. S4 B). Bar, 10 µm. (F) Depletion of CUL4B impacts H2A ubiquitylation and ZRF1 recruitment. Chromatin association assays of UV irradiated HEK293T cells treated with siRNAs (control, CUL4B ). De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A-ubiquitin and ZRF1 abundance was calculated. Values are given as mean ± SEM ( n = 3). (G) Tethering of ZRF1 to chromatin depends on DDB2 during NER. Chromatin association assays in control fibroblasts (GM15876) and XPE (DDB2) fibroblasts (GM01389) after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative RING1B and ZRF1 abundance was calculated. Values are given as mean ± SEM ( n = 3).

    Techniques Used: Irradiation, Western Blot, Binding Assay, Expressing, Isolation, Incubation, Transfection, Staining, Inhibition

    Dissection of E3 ligase functions in UV-mediated DNA damage repair. (A) Quantitative analysis of H2A-ubiquitylation levels. Immunoblots (as in B and Fig. S1, A and B ) were probed with histone H2A antibody. The intensities of H2A and H2A-ubiquitin bands were quantified by the ImageJ software. The graphs illustrate the relative H2A ubiquitylation calculated as (H2A ubiquitin)/(H2A + H2A ubiquitin), normalized to Ponceau staining intensity after knockdown of the respective proteins (H2A ubiquitin/H2A). Values are normalized to the value from nonirradiated cells and are given as mean ± SEM ( n = 4). (B) Monoubiquitylation of histone H2A at lysine 119 after UV irradiation is mainly catalyzed by RING1B. Chromatin association assays of control and RING1B knockdown HEK293T cells after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The specificity of the H2A-ubiquitin antibody was verified (Fig. S1 C). (C) Epistatic relationship of xpc-1 and spat-3. Wild-type nematodes (N2) or spat-3 mutants (VC31) were fed with either control or xpc-1 RNAi–producing bacteria. The relative viability was analyzed after UV irradiation (200 J/m 2 ). Values are given as mean ± SEM ( n = 3). (D) Impact of BMI-1 on RING1B-mediated H2A ubiquitylation after UV irradiation. Chromatin association assays of UV-irradiated HEK293T cells treated with siRNAs (control, BMI-1 ). De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. Relative intensities of H2A ubiquitin/H2A and RING1B abundance after BMI-1 depletion were measured. Values are given as mean ± SEM ( n = 4). (E) Epistatic relationship of RING1B and BMI-1 in response to UV irradiation. Relative colony formation potential of control or RING1B knockdown cell lines treated with siRNA was analyzed at different UV doses. Control cells were transfected with either control siRNA (control) or BMI-1 siRNA ( BMI-1 ). RING1B knockdown cell lines were transfected with either control siRNA ( RING1B ) or BMI-1 siRNA ( RING1B + BMI-1 ). Gene knockdown was confirmed by Western blots (not depicted). Values are given as mean ± SEM ( n = 9).
    Figure Legend Snippet: Dissection of E3 ligase functions in UV-mediated DNA damage repair. (A) Quantitative analysis of H2A-ubiquitylation levels. Immunoblots (as in B and Fig. S1, A and B ) were probed with histone H2A antibody. The intensities of H2A and H2A-ubiquitin bands were quantified by the ImageJ software. The graphs illustrate the relative H2A ubiquitylation calculated as (H2A ubiquitin)/(H2A + H2A ubiquitin), normalized to Ponceau staining intensity after knockdown of the respective proteins (H2A ubiquitin/H2A). Values are normalized to the value from nonirradiated cells and are given as mean ± SEM ( n = 4). (B) Monoubiquitylation of histone H2A at lysine 119 after UV irradiation is mainly catalyzed by RING1B. Chromatin association assays of control and RING1B knockdown HEK293T cells after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The specificity of the H2A-ubiquitin antibody was verified (Fig. S1 C). (C) Epistatic relationship of xpc-1 and spat-3. Wild-type nematodes (N2) or spat-3 mutants (VC31) were fed with either control or xpc-1 RNAi–producing bacteria. The relative viability was analyzed after UV irradiation (200 J/m 2 ). Values are given as mean ± SEM ( n = 3). (D) Impact of BMI-1 on RING1B-mediated H2A ubiquitylation after UV irradiation. Chromatin association assays of UV-irradiated HEK293T cells treated with siRNAs (control, BMI-1 ). De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. Relative intensities of H2A ubiquitin/H2A and RING1B abundance after BMI-1 depletion were measured. Values are given as mean ± SEM ( n = 4). (E) Epistatic relationship of RING1B and BMI-1 in response to UV irradiation. Relative colony formation potential of control or RING1B knockdown cell lines treated with siRNA was analyzed at different UV doses. Control cells were transfected with either control siRNA (control) or BMI-1 siRNA ( BMI-1 ). RING1B knockdown cell lines were transfected with either control siRNA ( RING1B ) or BMI-1 siRNA ( RING1B + BMI-1 ). Gene knockdown was confirmed by Western blots (not depicted). Values are given as mean ± SEM ( n = 9).

    Techniques Used: Dissection, Western Blot, Software, Staining, Irradiation, Transfection

    14) Product Images from "Histones bundle F-actin filaments and affect actin structure"

    Article Title: Histones bundle F-actin filaments and affect actin structure

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0183760

    TIRF microscopy revealed a change in morphology of histone-induced F-actin bundles in the presence of cofilin. In all experiments actin was polymerized by 1 mM MgCl 2 containing in TIRF imaging buffer. (A), Actin (1μM) polymerized in TIRF imaging buffer for 15 minutes forms unbundled long filaments. (B), Actin (1μM) copolymerized with 1 μM cofilin in TIRF imaging buffer for 15 minutes. More short filaments were detected in the sample due to the cofilin-induced severing. (C-D), Actin (1μM) copolymerized with 1 μM (14 μg/ml) H2A histone in TIRF imaging buffer for 15 (C) and 30 minutes (D), respectively. Thin, cable-like F-actin bundles were detected. (E-F), Actin (1μM) copolymerized with 1 μM (14 μg/ml) H2A histone and 1 μM cofilin in TIRF imaging buffer for 15 (E) and 30 minutes (F). Star-like F-actin structures were detected under these conditions. Scale bars: 10 μm. Presented data are representative of three independently taken microscope images. TIRF imaging buffer: 10 mM Hepes, 1 mM MgCl2, 50 mM KCl, 0.2 mM EGTA (pH 7.4) supplemented with 50 mM DTT, 0.2 mM ATP, 0.05 mg/ml casein, 20 mM glucose, 0.25 mg/ml glucose oxidase, 50 μM catalase, 0.5% methyl cellulose. TIRF microscopy and Alexa 484 SE labeling were carried out as described in MATERIALS and METHODS.
    Figure Legend Snippet: TIRF microscopy revealed a change in morphology of histone-induced F-actin bundles in the presence of cofilin. In all experiments actin was polymerized by 1 mM MgCl 2 containing in TIRF imaging buffer. (A), Actin (1μM) polymerized in TIRF imaging buffer for 15 minutes forms unbundled long filaments. (B), Actin (1μM) copolymerized with 1 μM cofilin in TIRF imaging buffer for 15 minutes. More short filaments were detected in the sample due to the cofilin-induced severing. (C-D), Actin (1μM) copolymerized with 1 μM (14 μg/ml) H2A histone in TIRF imaging buffer for 15 (C) and 30 minutes (D), respectively. Thin, cable-like F-actin bundles were detected. (E-F), Actin (1μM) copolymerized with 1 μM (14 μg/ml) H2A histone and 1 μM cofilin in TIRF imaging buffer for 15 (E) and 30 minutes (F). Star-like F-actin structures were detected under these conditions. Scale bars: 10 μm. Presented data are representative of three independently taken microscope images. TIRF imaging buffer: 10 mM Hepes, 1 mM MgCl2, 50 mM KCl, 0.2 mM EGTA (pH 7.4) supplemented with 50 mM DTT, 0.2 mM ATP, 0.05 mg/ml casein, 20 mM glucose, 0.25 mg/ml glucose oxidase, 50 μM catalase, 0.5% methyl cellulose. TIRF microscopy and Alexa 484 SE labeling were carried out as described in MATERIALS and METHODS.

    Techniques Used: Microscopy, Imaging, Labeling

    Polymerization of CaATP-G-actin by histone mixture and H2A histone followed by increase in pyrene fluorescence. (A) 5.25–126 μg/ml histone mixture, (B) 0.5–6 μM (7–84 μg/ml) H2A histone, or 2 mM MgCl 2 were added to pyrene labeled (10% labeling ratio) 4 μM CaATP-G-actin in pH 7.4 CaATP-G-buffer. (C), 21 μg/ml histone mixture was added to pyrene labeled (10% labeling ratio) 4 μM CaATP-G-actin in pH 6.5, 7.4 and 8.2 CaATP-G-buffer or 2 mM MgCl 2 was added in pH 6.5 CaATP-G-buffer. Fluorescence measurements were carried out as given in MATERIALS and METHODS. Presented data are representative of three independent experiments.
    Figure Legend Snippet: Polymerization of CaATP-G-actin by histone mixture and H2A histone followed by increase in pyrene fluorescence. (A) 5.25–126 μg/ml histone mixture, (B) 0.5–6 μM (7–84 μg/ml) H2A histone, or 2 mM MgCl 2 were added to pyrene labeled (10% labeling ratio) 4 μM CaATP-G-actin in pH 7.4 CaATP-G-buffer. (C), 21 μg/ml histone mixture was added to pyrene labeled (10% labeling ratio) 4 μM CaATP-G-actin in pH 6.5, 7.4 and 8.2 CaATP-G-buffer or 2 mM MgCl 2 was added in pH 6.5 CaATP-G-buffer. Fluorescence measurements were carried out as given in MATERIALS and METHODS. Presented data are representative of three independent experiments.

    Techniques Used: Fluorescence, Labeling

    Effect of cofilin on the sedimentation of 4 μM MgF-actin bundled by histone mixture or H2A histone. (A), 2–8 μM cofilin added to 4 μM F-actin bundled by 10.5 and 63 μg/ml histone mixture or (B) by 1 μM (14 μg/ml) and 3 μM (42 μg/ml) H2A histone. (C), 42 μg/ml histone mixture or 4 μM (56 μg/ml) H2A histone and 2.5 or 5 μM cofilin were added simultaneously to 4 μM F-actin. Samples were centrifuged at 20800xg for 8 min, supernatants run on SDS-PAGE and evaluated as described in MATERIALS and METHODS. The presented data are mean and standard deviation of three independent experiments. Insets: lanes of SDS-PAGE gels, representatives of three independent experiments, obtained from SDS-PAGE of low speed centrifugation supernatants.
    Figure Legend Snippet: Effect of cofilin on the sedimentation of 4 μM MgF-actin bundled by histone mixture or H2A histone. (A), 2–8 μM cofilin added to 4 μM F-actin bundled by 10.5 and 63 μg/ml histone mixture or (B) by 1 μM (14 μg/ml) and 3 μM (42 μg/ml) H2A histone. (C), 42 μg/ml histone mixture or 4 μM (56 μg/ml) H2A histone and 2.5 or 5 μM cofilin were added simultaneously to 4 μM F-actin. Samples were centrifuged at 20800xg for 8 min, supernatants run on SDS-PAGE and evaluated as described in MATERIALS and METHODS. The presented data are mean and standard deviation of three independent experiments. Insets: lanes of SDS-PAGE gels, representatives of three independent experiments, obtained from SDS-PAGE of low speed centrifugation supernatants.

    Techniques Used: Sedimentation, SDS Page, Standard Deviation, Centrifugation

    Effect of DNase1 on the light scattering and sedimentation of 4 μM MgF-actin bundled by histone mixture or H2A histone. (A) Effect of 9 μM DNase1 on the light scattering of 4 μM MgF-actin bundled by 63 μg/ml histone mixture or 3 μM (42 μg/ml) H2A histone. Light scattering change was followed as described in MATERIALS and METHODS. Presented data are representative of three independent experiments. (B). Effect of 2–15 μM DNase1 on the sedimentation of 4 μM MgF-actin bundled by 63 μg/ml histone mixture or 3 μM (42 μg/ml) H2A histone. The difference between the amount of actin sedimented following DNase1 treatment of histone mixture and H2A histone bundled actin is highly significant. Samples were centrifuged at 20800xg for 8 min, supernatants run on SDS-PAGE and evaluated as described in MATERIALS and METHODS. The presented data are mean and standard deviation of three independent experiments. Insets: actin lanes, representatives of three independent experiments, from SDS-PAGE of low speed centrifugation supernatants.
    Figure Legend Snippet: Effect of DNase1 on the light scattering and sedimentation of 4 μM MgF-actin bundled by histone mixture or H2A histone. (A) Effect of 9 μM DNase1 on the light scattering of 4 μM MgF-actin bundled by 63 μg/ml histone mixture or 3 μM (42 μg/ml) H2A histone. Light scattering change was followed as described in MATERIALS and METHODS. Presented data are representative of three independent experiments. (B). Effect of 2–15 μM DNase1 on the sedimentation of 4 μM MgF-actin bundled by 63 μg/ml histone mixture or 3 μM (42 μg/ml) H2A histone. The difference between the amount of actin sedimented following DNase1 treatment of histone mixture and H2A histone bundled actin is highly significant. Samples were centrifuged at 20800xg for 8 min, supernatants run on SDS-PAGE and evaluated as described in MATERIALS and METHODS. The presented data are mean and standard deviation of three independent experiments. Insets: actin lanes, representatives of three independent experiments, from SDS-PAGE of low speed centrifugation supernatants.

    Techniques Used: Sedimentation, SDS Page, Standard Deviation, Centrifugation

    Effect of 3 μM (42 μg/ml) H2A histone and 6 μM cofilin on the viscosity of 4 μM MgF-actin measured by Viscous Aqua fluorescence viscosity probe. Viscous Aqua in original Ursa BioScience vial was dissolved in 50 μl methanol then diluted 50 times in actin buffer and added to actin containing solutions in 1 to 50 ratio in pH 7.4 buffer. The fluorescence of the mixtures was measured as described in MATERIALS and METHODS. The fluorescence values (in artificial units, A.U.) of the samples at 492 nm emission maximum minus the fluorescence of the buffer are given in the figure. The data obtained were compared by statistical analysis and the significance of the differences was indicated. * = p
    Figure Legend Snippet: Effect of 3 μM (42 μg/ml) H2A histone and 6 μM cofilin on the viscosity of 4 μM MgF-actin measured by Viscous Aqua fluorescence viscosity probe. Viscous Aqua in original Ursa BioScience vial was dissolved in 50 μl methanol then diluted 50 times in actin buffer and added to actin containing solutions in 1 to 50 ratio in pH 7.4 buffer. The fluorescence of the mixtures was measured as described in MATERIALS and METHODS. The fluorescence values (in artificial units, A.U.) of the samples at 492 nm emission maximum minus the fluorescence of the buffer are given in the figure. The data obtained were compared by statistical analysis and the significance of the differences was indicated. * = p

    Techniques Used: Fluorescence

    Effect of cofilin on the light scattering of histone mixture or H2A histone bundled MgF-actin. 4x 2 μM cofilin was added to 4 μM MgF-actin bundled by 63 μg/ml histone mixture (A), or by 4 μM (56 μg/ml) H2A histone (B). Stars indicate addition of 2 μM cofilin. Light scattering change was followed as described in MATERIALS and METHODS. Presented data are representative of three independent experiments.
    Figure Legend Snippet: Effect of cofilin on the light scattering of histone mixture or H2A histone bundled MgF-actin. 4x 2 μM cofilin was added to 4 μM MgF-actin bundled by 63 μg/ml histone mixture (A), or by 4 μM (56 μg/ml) H2A histone (B). Stars indicate addition of 2 μM cofilin. Light scattering change was followed as described in MATERIALS and METHODS. Presented data are representative of three independent experiments.

    Techniques Used:

    Histone mixture and H2A histone induced bundle formation of Mg-F-actin followed by low speed centrifugation and by light scattering. 5.25–84 μg/ml histone mixture (A), or 1–4 μM (14–56 μg/ml) H2A histone (B), were added to 4 μM MgF-actin in pH7.4 F-buffer and centrifuged at low speed. Samples were centrifuged at 20,800xg for 8 min, supernatants run on SDS-PAGE and evaluated as described in MATERIALS and METHODS. The presented data are mean and standard deviation of three independent experiments. Insets: actin lanes, representatives of three independent experiments, from SDS-PAGE of low speed centrifugation supernatants. (C) 5.25–42 μg/ml histone mixture or (D) 1–4 μM (14–56 μg/ml) H2A histone were added to 4 μM MgF-actin in pH7.4 F-buffer and the light scattering change was followed as described in MATERIALS and METHODS. Presented data are representative of three independent experiments. All measurements were done at pH7.4 in F-buffer.
    Figure Legend Snippet: Histone mixture and H2A histone induced bundle formation of Mg-F-actin followed by low speed centrifugation and by light scattering. 5.25–84 μg/ml histone mixture (A), or 1–4 μM (14–56 μg/ml) H2A histone (B), were added to 4 μM MgF-actin in pH7.4 F-buffer and centrifuged at low speed. Samples were centrifuged at 20,800xg for 8 min, supernatants run on SDS-PAGE and evaluated as described in MATERIALS and METHODS. The presented data are mean and standard deviation of three independent experiments. Insets: actin lanes, representatives of three independent experiments, from SDS-PAGE of low speed centrifugation supernatants. (C) 5.25–42 μg/ml histone mixture or (D) 1–4 μM (14–56 μg/ml) H2A histone were added to 4 μM MgF-actin in pH7.4 F-buffer and the light scattering change was followed as described in MATERIALS and METHODS. Presented data are representative of three independent experiments. All measurements were done at pH7.4 in F-buffer.

    Techniques Used: Centrifugation, SDS Page, Standard Deviation

    Effect of DNA on the bundling of F-actin by histone. (A), Effect of 25–400 μg/ml DNA on the sedimentation of 4 μM MgF-actin bundled by 42 μg/ml histone mixture or 3 μM (42 μg/ml) H2A histone. (B), Effect of 0–200 μg/ml DNA digested by Staphylococcus aureus micrococcal DNase on the sedimentation of 4 μM MgF-actin bundled by 42 μg/ml histone mixture or by 3 μM (42 μg/ml) H2A histone. 4 μg/ml DNA was digested by 20 μg/ml micrococcal DNase at 37°C for 30 min. Samples were centrifuged at 20800xg for 8 min, supernatants run on SDS-PAGE and evaluated as described in MATERIALS and METHODS. The presented data are mean and standard deviation of three independent experiments. Insets: actin lanes, representatives of three independent experiments, from the SDS-PAGE of low speed centrifugation supernatants.
    Figure Legend Snippet: Effect of DNA on the bundling of F-actin by histone. (A), Effect of 25–400 μg/ml DNA on the sedimentation of 4 μM MgF-actin bundled by 42 μg/ml histone mixture or 3 μM (42 μg/ml) H2A histone. (B), Effect of 0–200 μg/ml DNA digested by Staphylococcus aureus micrococcal DNase on the sedimentation of 4 μM MgF-actin bundled by 42 μg/ml histone mixture or by 3 μM (42 μg/ml) H2A histone. 4 μg/ml DNA was digested by 20 μg/ml micrococcal DNase at 37°C for 30 min. Samples were centrifuged at 20800xg for 8 min, supernatants run on SDS-PAGE and evaluated as described in MATERIALS and METHODS. The presented data are mean and standard deviation of three independent experiments. Insets: actin lanes, representatives of three independent experiments, from the SDS-PAGE of low speed centrifugation supernatants.

    Techniques Used: Sedimentation, SDS Page, Standard Deviation, Centrifugation

    Effect of 0–400 mM NaCl on the sedimentation of 63 μg/ml histone mixture or 4 μM (56 μg/ml) H2A histone bundled 4 μM MgF-actin as measured by low speed centrifugation and light scattering. Sedimentation: (A), Bundling by histone mixture and H2A histone. Samples were centrifuged at 20800xg for 8 min, supernatants run on SDS-PAGE and evaluated as described in MATERIALS and METHODS. The presented data are mean and standard deviation of three independent experiments. Insets: actin lanes, representatives of three independent experiments, from the SDS-PAGE of supernatants after low speed centrifugation. Light scattering: (B), 4x100 mM NaCl was added to histone mixture bundled 4 μM MgF-actin, (C), 3x100 mM NaCl was added to H2A histone bundled 4 μM MgF-actin and the light scattering was measured. Asterisks* represent 100 mM NaCl addition. Light scattering change was followed as described in MATERIALS and METHODS. Presented data are representative of three independent experiments.
    Figure Legend Snippet: Effect of 0–400 mM NaCl on the sedimentation of 63 μg/ml histone mixture or 4 μM (56 μg/ml) H2A histone bundled 4 μM MgF-actin as measured by low speed centrifugation and light scattering. Sedimentation: (A), Bundling by histone mixture and H2A histone. Samples were centrifuged at 20800xg for 8 min, supernatants run on SDS-PAGE and evaluated as described in MATERIALS and METHODS. The presented data are mean and standard deviation of three independent experiments. Insets: actin lanes, representatives of three independent experiments, from the SDS-PAGE of supernatants after low speed centrifugation. Light scattering: (B), 4x100 mM NaCl was added to histone mixture bundled 4 μM MgF-actin, (C), 3x100 mM NaCl was added to H2A histone bundled 4 μM MgF-actin and the light scattering was measured. Asterisks* represent 100 mM NaCl addition. Light scattering change was followed as described in MATERIALS and METHODS. Presented data are representative of three independent experiments.

    Techniques Used: Sedimentation, Centrifugation, SDS Page, Standard Deviation

    Polymerization of CaATP-G-actin by histone mixture and H2A histone followed by high speed centrifugation was compared with the plateaus of the pyrene fluorescent measurements. (A) 5.25–63 μg/ml histone mixture, or (B) 0.5–4 μM (7–56 μg/ml) H2A histone was added to 4 μM CaATP-G-actin in pH7.4 CaATP-G-buffer. Samples were centrifuged at 129,151xg for 2h, supernatants run on SDS-PAGE and evaluated as described in MATERIALS and METHODS. Fig 2A, inset: SDS-PAGE, left, actin and histone mixture before centrifugation; right, molecular weight marker. Fig 2B, inset: actin lanes from the SDS-PAGE of supernatants after high speed centrifugation. All SDS-PAGE gels are representatives of three independent experiments. Actin sedimentation values were compared with plateaus of pyrene fluorescence upon addition of histone mixture (C) or H2A histone (D). Pyrene fluorescence values were taken from Fig 1 . Sedimentation data were taken from experiments presented in Fig 2A and B. The presented data are mean and standard deviation of three independent experiments.
    Figure Legend Snippet: Polymerization of CaATP-G-actin by histone mixture and H2A histone followed by high speed centrifugation was compared with the plateaus of the pyrene fluorescent measurements. (A) 5.25–63 μg/ml histone mixture, or (B) 0.5–4 μM (7–56 μg/ml) H2A histone was added to 4 μM CaATP-G-actin in pH7.4 CaATP-G-buffer. Samples were centrifuged at 129,151xg for 2h, supernatants run on SDS-PAGE and evaluated as described in MATERIALS and METHODS. Fig 2A, inset: SDS-PAGE, left, actin and histone mixture before centrifugation; right, molecular weight marker. Fig 2B, inset: actin lanes from the SDS-PAGE of supernatants after high speed centrifugation. All SDS-PAGE gels are representatives of three independent experiments. Actin sedimentation values were compared with plateaus of pyrene fluorescence upon addition of histone mixture (C) or H2A histone (D). Pyrene fluorescence values were taken from Fig 1 . Sedimentation data were taken from experiments presented in Fig 2A and B. The presented data are mean and standard deviation of three independent experiments.

    Techniques Used: Centrifugation, SDS Page, Molecular Weight, Marker, Sedimentation, Fluorescence, Standard Deviation

    15) Product Images from "Quantitative Proteomics to Characterize Specific Histone H2A Proteolysis in Chronic Lymphocytic Leukemia and the Myeloid THP-1 Cell Line"

    Article Title: Quantitative Proteomics to Characterize Specific Histone H2A Proteolysis in Chronic Lymphocytic Leukemia and the Myeloid THP-1 Cell Line

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms15069407

    Specific cH2A quantitation in myeloid cell lines. ( A ) Example of an MS spectrum of the AQUA 2 peptide if H2A V 114 clipping is absent ( left ) or present ( right ). The analysis of different cell lines confirms the myeloid characteristic of cH2A in the U-937, THP-1 and HL-60 cells. H2A clipping is not present in the investigated lymphatic cell lines and could not be unambiguously detected in the promyelocytic HL-60 cell line; ( B ) THP-1 cells stimulated with PMA show a transient H2A clipping pattern. Three biological replicates display the high variance. * 1: significant differences of %cH2A between T PMA0 and the other data points; * 2: the equivalent for T PMA60 . The %cH2A of synchronized stimulated and control THP-1 cells, presented respectively as squares and triangles, is lower than the %cH2A of non-synchronized cells.
    Figure Legend Snippet: Specific cH2A quantitation in myeloid cell lines. ( A ) Example of an MS spectrum of the AQUA 2 peptide if H2A V 114 clipping is absent ( left ) or present ( right ). The analysis of different cell lines confirms the myeloid characteristic of cH2A in the U-937, THP-1 and HL-60 cells. H2A clipping is not present in the investigated lymphatic cell lines and could not be unambiguously detected in the promyelocytic HL-60 cell line; ( B ) THP-1 cells stimulated with PMA show a transient H2A clipping pattern. Three biological replicates display the high variance. * 1: significant differences of %cH2A between T PMA0 and the other data points; * 2: the equivalent for T PMA60 . The %cH2A of synchronized stimulated and control THP-1 cells, presented respectively as squares and triangles, is lower than the %cH2A of non-synchronized cells.

    Techniques Used: Quantitation Assay, Mass Spectrometry

    Quantitative mass spectrometry and Western blot analysis on CLL samples revealed that histone H2A clipping is from myeloid origin. ( A ) The iTRAQ ratios of the cH2A peptide (normalized to the average of the H2A protein to compensate for morphological differences) hint towards an increased abundance in CLL compared to healthy B-cells. The individual log ratios of all six runs are presented as dots, the average ratios as the horizontal bars. Histone H2A (cH2A) is up-regulated in the samples of the leukemia pool compared to the samples of the healthy B-cells in all six runs; ( B ) The histoneextracts from 12 of the 36 samples (From left to right: patient samples UM + 8, 9, 10 and 11; M − 9, 10, 11, 12, 13 and 14; M + 5 and UM − 6). Western blot with an H2A antibody against the epitope depicted in Figure 1 detects the H2A variants H2Ax, Ubiquitinated H2A and macroH2A. The band under ubiquitinated H2A could not be identified. cH2A was only faintly detected, except for one sample ( * ); ( C ) Specific cH2A screening with AQUA peptides and flow cytometry data revealed the myeloid characteristic of the clipping. Left panel : %cH2A differs significantly ( p = 0.049) between CLL patients with a distinct mutational status (UM: 11× UM + 6× UM − , M: 14× M − 5× M + ); Middle panel : Although not significant ( p = 0.15), the %CD66b suggested a similar correlation with the mutational status; Right panel : Relation between %cH2a and %CD66b. Spearman’s Rho correlation between CD66b + and %cH2A was significant at the 0.01 level (Spearman’s Rho correlation coefficient: 0.439; p = 0.007; Data: Table S2b ).
    Figure Legend Snippet: Quantitative mass spectrometry and Western blot analysis on CLL samples revealed that histone H2A clipping is from myeloid origin. ( A ) The iTRAQ ratios of the cH2A peptide (normalized to the average of the H2A protein to compensate for morphological differences) hint towards an increased abundance in CLL compared to healthy B-cells. The individual log ratios of all six runs are presented as dots, the average ratios as the horizontal bars. Histone H2A (cH2A) is up-regulated in the samples of the leukemia pool compared to the samples of the healthy B-cells in all six runs; ( B ) The histoneextracts from 12 of the 36 samples (From left to right: patient samples UM + 8, 9, 10 and 11; M − 9, 10, 11, 12, 13 and 14; M + 5 and UM − 6). Western blot with an H2A antibody against the epitope depicted in Figure 1 detects the H2A variants H2Ax, Ubiquitinated H2A and macroH2A. The band under ubiquitinated H2A could not be identified. cH2A was only faintly detected, except for one sample ( * ); ( C ) Specific cH2A screening with AQUA peptides and flow cytometry data revealed the myeloid characteristic of the clipping. Left panel : %cH2A differs significantly ( p = 0.049) between CLL patients with a distinct mutational status (UM: 11× UM + 6× UM − , M: 14× M − 5× M + ); Middle panel : Although not significant ( p = 0.15), the %CD66b suggested a similar correlation with the mutational status; Right panel : Relation between %cH2a and %CD66b. Spearman’s Rho correlation between CD66b + and %cH2A was significant at the 0.01 level (Spearman’s Rho correlation coefficient: 0.439; p = 0.007; Data: Table S2b ).

    Techniques Used: Mass Spectrometry, Western Blot, Flow Cytometry, Cytometry

    Sequence of histone H2A. Sequence from Uniprot [ 25 ]. The VTIAQGGVLPNIQAV peptide ( m / z 740.4, charge 2+) was the only semi-tryptic peptide identified in all six runs. * indicates the clipping site after V 114 (here described as amino acid 115 since methionine, generated by the start codon, is the first amino acid in the Uniprot sequence); Bold underlined sequences are the same as the isotopically labeled absolute quantification (AQUA) peptides, used for the subsequent specific cH2A quantitation. The bold double underlined sequence is the Western blot epitope.
    Figure Legend Snippet: Sequence of histone H2A. Sequence from Uniprot [ 25 ]. The VTIAQGGVLPNIQAV peptide ( m / z 740.4, charge 2+) was the only semi-tryptic peptide identified in all six runs. * indicates the clipping site after V 114 (here described as amino acid 115 since methionine, generated by the start codon, is the first amino acid in the Uniprot sequence); Bold underlined sequences are the same as the isotopically labeled absolute quantification (AQUA) peptides, used for the subsequent specific cH2A quantitation. The bold double underlined sequence is the Western blot epitope.

    Techniques Used: Sequencing, Generated, Labeling, Quantitation Assay, Western Blot

    16) Product Images from "Recruitment of ubiquitin-activating enzyme UBA1 to DNA by poly(ADP-ribose) promotes ATR signalling"

    Article Title: Recruitment of ubiquitin-activating enzyme UBA1 to DNA by poly(ADP-ribose) promotes ATR signalling

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201800096

    Human UBA1 binds to pADPr chains. (A) Homo sapiens UBA1, H2A, and BSA were spotted on a nitrocellulose membrane, incubated with [ 32 P]-labelled pADPr, washed, and exposed to autoradiography. (B) Purified H2A, BSA, H. sapiens UBA1 (UBA1), S. cerevisiae Uba1 (ScUba1), and H. sapiens UBA6 (UBA6) were spotted on nitrocellulose membrane and incubated with purified pADPr chains. The retention of pADPr on the membrane was revealed using an anti-pADPr antibody. (C) Increasing amounts of purified H. sapiens UBA1 (UBA1) and S. cerevisiae Uba1 (ScUba1) were spotted on nitrocellulose membrane and incubated with purified pADPr chains. The retention of pADPr on the membrane was revealed using an anti-pADPr antibody. (D) Left panel: schematic representation of UBA1 with its functional domains and the six purified overlapping fragments of UBA1. Right panel: Purified MBP-UBA1 fragments resolved by PAGE and stained by Coomassie Brilliant Blue. (E) UBA1 fragments (10 pM) were spotted on nitrocellulose membrane incubated with purified pADPr chains. Immobilised pADPr was revealed using an anti-pADPr antibody. (F) Left panel: experimental scheme. MBP UBA1 (571–800) was incubated with pADPr polymers, captured on an amylose resin, washed, and eluted with maltose. Eluted MBP UBA1 (571–800) was resolved by PAGE and revealed via anti-MBP immunoblotting. Eluted pADPr was spotted on a nitrocellulose membrane and revealed using an anti-pADPr antibody. IAD, inactive adenylation domain; FCCH, first catalytic cysteine half domain; AAD, active adenylation domain; SCCH, second catalytic cysteine half domain.
    Figure Legend Snippet: Human UBA1 binds to pADPr chains. (A) Homo sapiens UBA1, H2A, and BSA were spotted on a nitrocellulose membrane, incubated with [ 32 P]-labelled pADPr, washed, and exposed to autoradiography. (B) Purified H2A, BSA, H. sapiens UBA1 (UBA1), S. cerevisiae Uba1 (ScUba1), and H. sapiens UBA6 (UBA6) were spotted on nitrocellulose membrane and incubated with purified pADPr chains. The retention of pADPr on the membrane was revealed using an anti-pADPr antibody. (C) Increasing amounts of purified H. sapiens UBA1 (UBA1) and S. cerevisiae Uba1 (ScUba1) were spotted on nitrocellulose membrane and incubated with purified pADPr chains. The retention of pADPr on the membrane was revealed using an anti-pADPr antibody. (D) Left panel: schematic representation of UBA1 with its functional domains and the six purified overlapping fragments of UBA1. Right panel: Purified MBP-UBA1 fragments resolved by PAGE and stained by Coomassie Brilliant Blue. (E) UBA1 fragments (10 pM) were spotted on nitrocellulose membrane incubated with purified pADPr chains. Immobilised pADPr was revealed using an anti-pADPr antibody. (F) Left panel: experimental scheme. MBP UBA1 (571–800) was incubated with pADPr polymers, captured on an amylose resin, washed, and eluted with maltose. Eluted MBP UBA1 (571–800) was resolved by PAGE and revealed via anti-MBP immunoblotting. Eluted pADPr was spotted on a nitrocellulose membrane and revealed using an anti-pADPr antibody. IAD, inactive adenylation domain; FCCH, first catalytic cysteine half domain; AAD, active adenylation domain; SCCH, second catalytic cysteine half domain.

    Techniques Used: Incubation, Autoradiography, Purification, Functional Assay, Polyacrylamide Gel Electrophoresis, Staining

    Related Articles

    Incubation:

    Article Title: Bub1-Mediated Adaptation of the Spindle Checkpoint
    Article Snippet: .. The volume was reduced to 50 µL, and the solution was incubated with 100 µM ATP, 0.2 µCi [gamma-32 P]ATP and substrate (10 µg histone H3 [USBiological, Swampscott, MA]; 1 µg histone H2A human, recombinant [New England BioLabs, MA]; 1 µg histone H1 [Upstate Biotechnology, Lake Placid, NY]; 1 µg BUB1_400-700-MBP and BUB1-T566A_400-700-MBP) at 30°C for 20 min. .. The reaction was stopped by adding SDS loading buffer, and the protein were then separated by SDS-PAGE, stained with Coomassie Brilliant Blue, and analyzed by autoradiography.

    Recombinant:

    Article Title: Probing the catalytic functions of Bub1 kinase using the small molecule inhibitors BAY-320 and BAY-524
    Article Snippet: .. Kinase reactions were carried out at 30°C in kinase buffer in the presence of 100 μM ATP, 5 μCi γ-32 P-ATP, 1 μg recombinant histone H2A (NEB, Frankfurt am Main, Germany) as substrate, and serial dilutions of Bub1 inhibitors. ..

    Article Title: Structure Change from β-Strand and Turn to α-Helix in Histone H2A-H2B Induced by DNA Damage Response
    Article Snippet: .. Bands between 25 and 40 kDa were also observed even in the case of recombinant H2A ( ). .. Therefore, these bands would originate from some kind of complex of H2A and its variants that formed during the SDS-PAGE analyses, because no aggregation was observed in the DLS measurements as mentioned above.

    Article Title: Bub1-Mediated Adaptation of the Spindle Checkpoint
    Article Snippet: .. The volume was reduced to 50 µL, and the solution was incubated with 100 µM ATP, 0.2 µCi [gamma-32 P]ATP and substrate (10 µg histone H3 [USBiological, Swampscott, MA]; 1 µg histone H2A human, recombinant [New England BioLabs, MA]; 1 µg histone H1 [Upstate Biotechnology, Lake Placid, NY]; 1 µg BUB1_400-700-MBP and BUB1-T566A_400-700-MBP) at 30°C for 20 min. .. The reaction was stopped by adding SDS loading buffer, and the protein were then separated by SDS-PAGE, stained with Coomassie Brilliant Blue, and analyzed by autoradiography.

    Article Title: Mammalian Protein Arginine Methyltransferase 7 (PRMT7) Specifically Targets RXR Sites in Lysine- and Arginine-rich Regions *
    Article Snippet: .. Recombinant human histone H2A (GenBankTM accession number ), H2B (GenBankTM accession number ), H3.3 (GenBankTM accession number ), and H4 (GenBankTM accession number ) were purchased from New England Biolabs as 1 mg/ml solutions in 20 m m sodium phosphate, 300 m m NaCl, and 1 m m EDTA. ..

    Article Title: Breaching peripheral tolerance promotes the production of HIV-1–neutralizing antibodies
    Article Snippet: .. For Histone H2A- and H2B-reactive IgM ELISAs, plates were coated with 1 µg/ml of human recombinant histone H2A or H2B (New England Biolabs) in PBS pH 7.4. ..

    Article Title: Proteolytic Histone Modification by Mast Cell Tryptase, a Serglycin Proteoglycan-dependent Secretory Granule Protease *
    Article Snippet: .. Recombinant human histone H2A, H2B, H3.1, and H4 were from New England Biolabs (Ipswich, MA). .. LysoTracker Red DND-99 was from Life Technologies.

    In Vitro:

    Article Title: Elevated H3K79 homocysteinylation causes abnormal gene expression during neural development and subsequent neural tube defects
    Article Snippet: .. HTL treatment in vitro Purified histones, including H2a (M2502S, NEB), H2b (M2505S, NEB), H3 (M2503S, NEB) and H4 (M2504S, NEB) were selected. ..

    Purification:

    Article Title: Elevated H3K79 homocysteinylation causes abnormal gene expression during neural development and subsequent neural tube defects
    Article Snippet: .. HTL treatment in vitro Purified histones, including H2a (M2502S, NEB), H2b (M2505S, NEB), H3 (M2503S, NEB) and H4 (M2504S, NEB) were selected. ..

    Article Title: Recruitment of ubiquitin-activating enzyme UBA1 to DNA by poly(ADP-ribose) promotes ATR signalling
    Article Snippet: .. Purified histone H2A (#M2502S) was obtained from New England Biolabs. .. Radioactive pADPr binding assay Radioactive pADPr binding assay was performed as described by , with some modifications.

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    New England Biolabs histone h2a
    Histone <t>H2A-reactive</t> IgM monoclonal antibodies, isolated from B6.Sle123 mice, neutralize tier 2 strains of HIV-1. Hybridomas were generated from splenocytes isolated from B6.Sle123 mice that displayed serum tier 2 HIV-1 neutralization ( n = 2). (A) Purified monoclonal IgM and IgG antibodies ( n = 8) were tested for HIV-1 neutralization against 4 strains of HIV-1 and reported as IC 50 (the concentration of antibody required for 50% neutralization). (B) Specificities of the monoclonal antibodies were tested using ELISA against the histone H2A, histone H2B, and chromatin nuclear antigens in addition to HIV-1 gp140 (YU2) Env and the CD4bs (RSC3) epitope, as well as a CD4bs-negative control (ΔRSC3). The two neutralizing IgM mAbs (P4E4 and O4C5) are shown with red symbols, and lines and non-neutralizing mAbs shown with solid black symbols and lines for IgM and gray symbols and dashed lines for IgG. IgM and IgG control antibodies were used to determine the background of the assay and for which ODs above this line were considered positive. Data are representative from three independent experiments.
    Histone H2a, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Histone H2A-reactive IgM monoclonal antibodies, isolated from B6.Sle123 mice, neutralize tier 2 strains of HIV-1. Hybridomas were generated from splenocytes isolated from B6.Sle123 mice that displayed serum tier 2 HIV-1 neutralization ( n = 2). (A) Purified monoclonal IgM and IgG antibodies ( n = 8) were tested for HIV-1 neutralization against 4 strains of HIV-1 and reported as IC 50 (the concentration of antibody required for 50% neutralization). (B) Specificities of the monoclonal antibodies were tested using ELISA against the histone H2A, histone H2B, and chromatin nuclear antigens in addition to HIV-1 gp140 (YU2) Env and the CD4bs (RSC3) epitope, as well as a CD4bs-negative control (ΔRSC3). The two neutralizing IgM mAbs (P4E4 and O4C5) are shown with red symbols, and lines and non-neutralizing mAbs shown with solid black symbols and lines for IgM and gray symbols and dashed lines for IgG. IgM and IgG control antibodies were used to determine the background of the assay and for which ODs above this line were considered positive. Data are representative from three independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Breaching peripheral tolerance promotes the production of HIV-1–neutralizing antibodies

    doi: 10.1084/jem.20161190

    Figure Lengend Snippet: Histone H2A-reactive IgM monoclonal antibodies, isolated from B6.Sle123 mice, neutralize tier 2 strains of HIV-1. Hybridomas were generated from splenocytes isolated from B6.Sle123 mice that displayed serum tier 2 HIV-1 neutralization ( n = 2). (A) Purified monoclonal IgM and IgG antibodies ( n = 8) were tested for HIV-1 neutralization against 4 strains of HIV-1 and reported as IC 50 (the concentration of antibody required for 50% neutralization). (B) Specificities of the monoclonal antibodies were tested using ELISA against the histone H2A, histone H2B, and chromatin nuclear antigens in addition to HIV-1 gp140 (YU2) Env and the CD4bs (RSC3) epitope, as well as a CD4bs-negative control (ΔRSC3). The two neutralizing IgM mAbs (P4E4 and O4C5) are shown with red symbols, and lines and non-neutralizing mAbs shown with solid black symbols and lines for IgM and gray symbols and dashed lines for IgG. IgM and IgG control antibodies were used to determine the background of the assay and for which ODs above this line were considered positive. Data are representative from three independent experiments.

    Article Snippet: For Histone H2A- and H2B-reactive IgM ELISAs, plates were coated with 1 µg/ml of human recombinant histone H2A or H2B (New England Biolabs) in PBS pH 7.4.

    Techniques: Isolation, Mouse Assay, Generated, Neutralization, Purification, Concentration Assay, Enzyme-linked Immunosorbent Assay, Negative Control

    Elevated IgM anti-histone H2A titers correlate with tier 2 HIV-1 neutralization by pristane treated wild-type C57BL/6 mice. Total serum concentrations of (A) IgM, (B) IgG, and (C) relative titers of serum IgM anti-H2A are shown for naive (open circles) B6 mice or B6 mice treated 30 d with pristane only (gray circles) and subsequently immunized 2X or 3X (black circles) with alum alone or Env + alum. Serum from individual B6 mice (regardless of treatment with pristane alone, alum alone, or Env + alum) were separated based on neutralization of ≥1 tier 2 HIV-1 strains and measured for (D) IgM anti-H2A or (E) IgM anti-H2B relative titers. Mice neutralizing only tier 1 strains were included in the tier 2 nonneutralizer group (mostly 3X). Each symbol represents measurements for one mouse, and all data are plotted as the arithmetic mean ± SEM. All P-values were calculated using Student’s t test assuming unequal variances. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Breaching peripheral tolerance promotes the production of HIV-1–neutralizing antibodies

    doi: 10.1084/jem.20161190

    Figure Lengend Snippet: Elevated IgM anti-histone H2A titers correlate with tier 2 HIV-1 neutralization by pristane treated wild-type C57BL/6 mice. Total serum concentrations of (A) IgM, (B) IgG, and (C) relative titers of serum IgM anti-H2A are shown for naive (open circles) B6 mice or B6 mice treated 30 d with pristane only (gray circles) and subsequently immunized 2X or 3X (black circles) with alum alone or Env + alum. Serum from individual B6 mice (regardless of treatment with pristane alone, alum alone, or Env + alum) were separated based on neutralization of ≥1 tier 2 HIV-1 strains and measured for (D) IgM anti-H2A or (E) IgM anti-H2B relative titers. Mice neutralizing only tier 1 strains were included in the tier 2 nonneutralizer group (mostly 3X). Each symbol represents measurements for one mouse, and all data are plotted as the arithmetic mean ± SEM. All P-values were calculated using Student’s t test assuming unequal variances. *, P

    Article Snippet: For Histone H2A- and H2B-reactive IgM ELISAs, plates were coated with 1 µg/ml of human recombinant histone H2A or H2B (New England Biolabs) in PBS pH 7.4.

    Techniques: Neutralization, Mouse Assay

    B6.Sle123 HIV-1 neutralizers harbor elevated levels of IgM anti-histone H2A. (A) Sera from B6 (open), B6.Sle123 nonneutralizers (gray), and neutralizers (black) were interrogated with an autoantigen array and results for the IgM reactive with the indicated anti-DNA antigens (top left), RNA-binding proteins (bottom left), and anti-histone antigens (top right), as well as IgG anti-histone antigens (bottom right) are shown. Relative serum titers of (B) IgM anti-H2A (left) and anti-H2B (right) measured by ELISA in B6.Sle123 neutralizers and nonneutralizers. Each symbol represents measurements for one mouse, and all data are plotted as the arithmetic mean ± SEM. P-values were calculated using the Mann-Whitney nonparametric test; *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Breaching peripheral tolerance promotes the production of HIV-1–neutralizing antibodies

    doi: 10.1084/jem.20161190

    Figure Lengend Snippet: B6.Sle123 HIV-1 neutralizers harbor elevated levels of IgM anti-histone H2A. (A) Sera from B6 (open), B6.Sle123 nonneutralizers (gray), and neutralizers (black) were interrogated with an autoantigen array and results for the IgM reactive with the indicated anti-DNA antigens (top left), RNA-binding proteins (bottom left), and anti-histone antigens (top right), as well as IgG anti-histone antigens (bottom right) are shown. Relative serum titers of (B) IgM anti-H2A (left) and anti-H2B (right) measured by ELISA in B6.Sle123 neutralizers and nonneutralizers. Each symbol represents measurements for one mouse, and all data are plotted as the arithmetic mean ± SEM. P-values were calculated using the Mann-Whitney nonparametric test; *, P

    Article Snippet: For Histone H2A- and H2B-reactive IgM ELISAs, plates were coated with 1 µg/ml of human recombinant histone H2A or H2B (New England Biolabs) in PBS pH 7.4.

    Techniques: RNA Binding Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    ( a ) Temperature dependence of the CD intensity at 222 nm of H2A-H2B extracted from core histones in unirradiated (Unirrad., open circle ) and x-irradiated (Irrad., solid circle ) cells. Inset: magnification of the unirradiated sample between 320

    Journal: Biophysical Journal

    Article Title: Structure Change from β-Strand and Turn to α-Helix in Histone H2A-H2B Induced by DNA Damage Response

    doi: 10.1016/j.bpj.2016.06.002

    Figure Lengend Snippet: ( a ) Temperature dependence of the CD intensity at 222 nm of H2A-H2B extracted from core histones in unirradiated (Unirrad., open circle ) and x-irradiated (Irrad., solid circle ) cells. Inset: magnification of the unirradiated sample between 320

    Article Snippet: Bands between 25 and 40 kDa were also observed even in the case of recombinant H2A ( ).

    Techniques: Irradiation

    Thermodynamic parameters of H2A-H2B

    Journal: Biophysical Journal

    Article Title: Structure Change from β-Strand and Turn to α-Helix in Histone H2A-H2B Induced by DNA Damage Response

    doi: 10.1016/j.bpj.2016.06.002

    Figure Lengend Snippet: Thermodynamic parameters of H2A-H2B

    Article Snippet: Bands between 25 and 40 kDa were also observed even in the case of recombinant H2A ( ).

    Techniques:

    ( a–c ) CD spectra of H2A-H2B extracted from core histones in unirradiated (Unirrad., black ) and x-irradiated (Irrad., red ) cells, measured at ( a ) 294, ( b ) 310, and ( c ) 330 K. In ( a ), a CD spectrum of H2A-H2B irradiated with 40 Gy x-rays

    Journal: Biophysical Journal

    Article Title: Structure Change from β-Strand and Turn to α-Helix in Histone H2A-H2B Induced by DNA Damage Response

    doi: 10.1016/j.bpj.2016.06.002

    Figure Lengend Snippet: ( a–c ) CD spectra of H2A-H2B extracted from core histones in unirradiated (Unirrad., black ) and x-irradiated (Irrad., red ) cells, measured at ( a ) 294, ( b ) 310, and ( c ) 330 K. In ( a ), a CD spectrum of H2A-H2B irradiated with 40 Gy x-rays

    Article Snippet: Bands between 25 and 40 kDa were also observed even in the case of recombinant H2A ( ).

    Techniques: Irradiation

    ( a–d ) Temperature dependence of the contents of ( a ) α -helix, ( b ) β -strand, ( c ) turn, and ( d ) other structures of H2A-H2B extracted from core histones in unirradiated (Unirrad., open circle ) and x-irradiated (Irrad., solid

    Journal: Biophysical Journal

    Article Title: Structure Change from β-Strand and Turn to α-Helix in Histone H2A-H2B Induced by DNA Damage Response

    doi: 10.1016/j.bpj.2016.06.002

    Figure Lengend Snippet: ( a–d ) Temperature dependence of the contents of ( a ) α -helix, ( b ) β -strand, ( c ) turn, and ( d ) other structures of H2A-H2B extracted from core histones in unirradiated (Unirrad., open circle ) and x-irradiated (Irrad., solid

    Article Snippet: Bands between 25 and 40 kDa were also observed even in the case of recombinant H2A ( ).

    Techniques: Irradiation

    ( a ) SDS-PAGE analysis of H2A-H2B extracted from core histones in unirradiated cells and 40 Gy irradiated cells after 30 min incubation. The lane labeled M refers to the standard molecular weight marker (XL-Ladder (low range); APRO Life

    Journal: Biophysical Journal

    Article Title: Structure Change from β-Strand and Turn to α-Helix in Histone H2A-H2B Induced by DNA Damage Response

    doi: 10.1016/j.bpj.2016.06.002

    Figure Lengend Snippet: ( a ) SDS-PAGE analysis of H2A-H2B extracted from core histones in unirradiated cells and 40 Gy irradiated cells after 30 min incubation. The lane labeled M refers to the standard molecular weight marker (XL-Ladder (low range); APRO Life

    Article Snippet: Bands between 25 and 40 kDa were also observed even in the case of recombinant H2A ( ).

    Techniques: SDS Page, Irradiation, Incubation, Labeling, Molecular Weight, Marker

    BAY-320 and BAY-524 inhibit Bub1 kinase. ( A, B ) BAY-320 and BAY-524 treatment coordinately reduces histone H2A-T120 phosphorylation as well as Aurora B centromere/KT binding, until maximal Bub1 inhibition is reached at 10 μM. Asynchronous cultures of HeLa S3 (left panels) and RPE1 cells (right panels) were treated with the proteasomal inhibitor MG132 for 2 hr, followed by the addition of 3.3 μM nocodazole and increasing doses of BAY-320 ( A ) or BAY-524 ( B ) for 1 hr. The cells were fixed and analyzed by immunofluorescence microscopy (IFM). Scatter plots show centromere/KT levels of pT120-H2A and Aurora B (n = 19–28 cells per condition). Bars represent mean values. ( C ) Untreated HeLa cells (red) or HeLa cells treated with nocodazole for 16 hr, followed by various concentrations of BAY-320 (green) or solvent (black) for 1 hr, were fixed and analyzed by quantitative in-cell western. Plot shows total pT120-H2A signal intensity. Grey area highlights the concentration range between 3 and 10 μM. The IC 50 (reflecting the inhibition of Bub1 kinase activity compared to control and normalized to cell number) was determined to be 379 +/- 156 nM. DOI: http://dx.doi.org/10.7554/eLife.12187.004

    Journal: eLife

    Article Title: Probing the catalytic functions of Bub1 kinase using the small molecule inhibitors BAY-320 and BAY-524

    doi: 10.7554/eLife.12187

    Figure Lengend Snippet: BAY-320 and BAY-524 inhibit Bub1 kinase. ( A, B ) BAY-320 and BAY-524 treatment coordinately reduces histone H2A-T120 phosphorylation as well as Aurora B centromere/KT binding, until maximal Bub1 inhibition is reached at 10 μM. Asynchronous cultures of HeLa S3 (left panels) and RPE1 cells (right panels) were treated with the proteasomal inhibitor MG132 for 2 hr, followed by the addition of 3.3 μM nocodazole and increasing doses of BAY-320 ( A ) or BAY-524 ( B ) for 1 hr. The cells were fixed and analyzed by immunofluorescence microscopy (IFM). Scatter plots show centromere/KT levels of pT120-H2A and Aurora B (n = 19–28 cells per condition). Bars represent mean values. ( C ) Untreated HeLa cells (red) or HeLa cells treated with nocodazole for 16 hr, followed by various concentrations of BAY-320 (green) or solvent (black) for 1 hr, were fixed and analyzed by quantitative in-cell western. Plot shows total pT120-H2A signal intensity. Grey area highlights the concentration range between 3 and 10 μM. The IC 50 (reflecting the inhibition of Bub1 kinase activity compared to control and normalized to cell number) was determined to be 379 +/- 156 nM. DOI: http://dx.doi.org/10.7554/eLife.12187.004

    Article Snippet: Kinase reactions were carried out at 30°C in kinase buffer in the presence of 100 μM ATP, 5 μCi γ-32 P-ATP, 1 μg recombinant histone H2A (NEB, Frankfurt am Main, Germany) as substrate, and serial dilutions of Bub1 inhibitors.

    Techniques: Binding Assay, Inhibition, Immunofluorescence, Microscopy, In-Cell ELISA, Concentration Assay, Activity Assay

    The processing of histones in viable and apoptotic mast cells is dependent on tryptase and serglycin. WT, serglycin −/− , and mMCP-6 −/− mast cells were treated with cytotoxic agent (CHX) or left untreated. In addition, WT cells were incubated in the presence of both cytotoxic agent and a general serine protease inhibitor (Pefabloc) as indicated. At the time points indicated, total cell extracts (corresponding to 0.5 × 10 6 cells/sample) were prepared and subjected to immunoblot analysis for core histones H2A, H2B, H3, and H4. β-Actin was used as loading control.

    Journal: The Journal of Biological Chemistry

    Article Title: Proteolytic Histone Modification by Mast Cell Tryptase, a Serglycin Proteoglycan-dependent Secretory Granule Protease *

    doi: 10.1074/jbc.M113.546895

    Figure Lengend Snippet: The processing of histones in viable and apoptotic mast cells is dependent on tryptase and serglycin. WT, serglycin −/− , and mMCP-6 −/− mast cells were treated with cytotoxic agent (CHX) or left untreated. In addition, WT cells were incubated in the presence of both cytotoxic agent and a general serine protease inhibitor (Pefabloc) as indicated. At the time points indicated, total cell extracts (corresponding to 0.5 × 10 6 cells/sample) were prepared and subjected to immunoblot analysis for core histones H2A, H2B, H3, and H4. β-Actin was used as loading control.

    Article Snippet: Recombinant human histone H2A, H2B, H3.1, and H4 were from New England Biolabs (Ipswich, MA).

    Techniques: Incubation, Protease Inhibitor