human histone h4  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95

    Structured Review

    New England Biolabs human histone h4
    Human Histone H4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human histone h4/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human histone h4 - by Bioz Stars, 2023-01
    95/100 stars

    Images

    human histone h4  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95

    Structured Review

    New England Biolabs human histone h4
    Human Histone H4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human histone h4/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human histone h4 - by Bioz Stars, 2023-01
    95/100 stars

    Images

    recombinant human histone h4  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95

    Structured Review

    New England Biolabs recombinant human histone h4
    A. Crosslinking <t>of</t> <t>H3–H4</t> . Histones H3 and H4 were incubated for the indicated minutes and then crosslinked with formaldehyde before separation on a 15% SDS/PAGE. The blot was probed separately with anti-H3 and anti-H4. B. Effect of Asf1 and TLK1B on H3–H4 dimer and tetramer formation. Reactions containing the indicated combinations of H3, H4, Asf1, and TLK1B (all in equivalent amount) were crosslinked as described in Methods and immunoblotted for H4 or H3. C. Western blots for Asf1 and TLK1B. The indicated reactions as in panel B were run in duplicate lanes and immunoblotted for Asf1, H3, or TLK1B (the gel was run for a longer time than in panel B to separate the larger proteins). For antibody controls, lane 1 contained only Asf1, and lane 7 contained only TLK1B. The positions of the cross-linked complexes identified by mobility and immunoreactivity are indicated. D. TLK1B and Asf1 bind each other stoichiometrically. Reactions contained 5 μg of GST-TLK1B and varying amounts of Asf1, as indicated. After 10 min at room temperature, the samples were adsorbed on GSH-Sepharose and analyzed for bound and unbound fractions after separation on a 10% SDS/PAGE, which was stained with Coomassie blue. E. Interaction of TLK1B and Asf1 and the effect of ATP. GST-TLK1B and Asf1 (1 μg each) were incubated for 10 min at room temperature with and without 10 or 100 μM ATP, before analysis by GSH-Sepharose pull-down. The 10% SDS/PAGE gel was stained with Coomassie blue. F. MNase digestion of pBluescript assembled into pseudo-nucleosomes. In the left panel, naked supercoiled plasmid was digested with MNase for the indicated time. In the right panel, the plasmid was first incubated with equimolar H3 and H4 in high salt and then step-dialyzed as described in , before MNase digestion. The DNA was re-extracted from the reactions with Geneclean and run on a 1.5% agarose/TAE gel. The resulting ~120 bp ladder (a bit shorter than the repetitive 146 bp of nucleosomal DNA) is indicative of formation of a chromatinized template. The positions of the bands of a 100-bp ladder (GenRuler, Fermentas) are indicated.
    Recombinant Human Histone H4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant human histone h4/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant human histone h4 - by Bioz Stars, 2023-01
    95/100 stars

    Images

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

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

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-2-128

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

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

    m2504s  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95

    Structured Review

    New England Biolabs m2504s
    KEY RESOURCES TABLE
    M2504s, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m2504s/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    m2504s - by Bioz Stars, 2023-01
    95/100 stars

    Images

    1) Product Images from "Induction of broadly reactive influenza antibodies increases susceptibility to autoimmunity"

    Article Title: Induction of broadly reactive influenza antibodies increases susceptibility to autoimmunity

    Journal: Cell reports

    doi: 10.1016/j.celrep.2022.110482

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Stripping Membranes, Microarray, Software, Imaging

    h4 histone  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95

    Structured Review

    New England Biolabs h4 histone
    KEY RESOURCES TABLE
    H4 Histone, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/h4 histone/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    h4 histone - by Bioz Stars, 2023-01
    95/100 stars

    Images

    1) Product Images from "Induction of broadly reactive influenza antibodies increases susceptibility to autoimmunity"

    Article Title: Induction of broadly reactive influenza antibodies increases susceptibility to autoimmunity

    Journal: Cell reports

    doi: 10.1016/j.celrep.2022.110482

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Stripping Membranes, Microarray, Software, Imaging

    h4  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95

    Structured Review

    New England Biolabs h4
    H4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/h4/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    h4 - by Bioz Stars, 2023-01
    95/100 stars

    Images

    recombinant human histone h4  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95

    Structured Review

    New England Biolabs recombinant human histone h4
    (A) Detection of unbound 100 bp DNA ladder by TBE ethidium bromide-stained gel in supernatants (left lane) on GST-SET8 domains or mutant (M) using GST-pulldown assays (top, left side). Asterisks are representing shift of DNA on GST-SET8 157-352 amino acid protein or GST-SET8 full-length protein (FL) beads. Ponceau stain represents the amount of GST beads constructs used for the GST-pulldown (bottom, left side). (B) Different concentrations of recombinant full-length SET8 protein binding to DNA using EMSA to determine the equilibrium dissociation constant (Kd). (C) Detection of unbound mononucleosome by TBE ethidium bromide-stained gel in supernatants on GST beads versus GST-SET8 domains or mutant (M) beads using GST-pulldown assays. (D) Detection of unbound DNA (left side) or unbound mononucleosome (right side) by TBE ethidium bromide-stained gel in supernatants (top) on GST beads versus GST-SET8 FL beads using GST-pulldown assays. GST or GST-SET8 FL beads were poly ADP-ribosylated or not (with or without NAD) using full-length recombinant PARP1 as demonstrated by western blot using anti-ADP ribose antibody (middle). Ponceau stain gel (bottom) represents the amount of GST bead constructs used for the GST-pulldown and the ADP-ribosylation western blot analysis. (E) SET8 histone methyltransferase assay on full-length recombinant histone <t>H4</t> using full-length recombinant SET8 in presence or absence of activated full-length recombinant PARP1.
    Recombinant Human Histone H4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant human histone h4/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant human histone h4 - by Bioz Stars, 2023-01
    95/100 stars

    Images

    1) Product Images from "Poly ADP-ribosylation of SET8 leads to aberrant H4K20 methylation domains in mammalian cells"

    Article Title: Poly ADP-ribosylation of SET8 leads to aberrant H4K20 methylation domains in mammalian cells

    Journal: bioRxiv

    doi: 10.1101/2021.11.13.468478

    (A) Detection of unbound 100 bp DNA ladder by TBE ethidium bromide-stained gel in supernatants (left lane) on GST-SET8 domains or mutant (M) using GST-pulldown assays (top, left side). Asterisks are representing shift of DNA on GST-SET8 157-352 amino acid protein or GST-SET8 full-length protein (FL) beads. Ponceau stain represents the amount of GST beads constructs used for the GST-pulldown (bottom, left side). (B) Different concentrations of recombinant full-length SET8 protein binding to DNA using EMSA to determine the equilibrium dissociation constant (Kd). (C) Detection of unbound mononucleosome by TBE ethidium bromide-stained gel in supernatants on GST beads versus GST-SET8 domains or mutant (M) beads using GST-pulldown assays. (D) Detection of unbound DNA (left side) or unbound mononucleosome (right side) by TBE ethidium bromide-stained gel in supernatants (top) on GST beads versus GST-SET8 FL beads using GST-pulldown assays. GST or GST-SET8 FL beads were poly ADP-ribosylated or not (with or without NAD) using full-length recombinant PARP1 as demonstrated by western blot using anti-ADP ribose antibody (middle). Ponceau stain gel (bottom) represents the amount of GST bead constructs used for the GST-pulldown and the ADP-ribosylation western blot analysis. (E) SET8 histone methyltransferase assay on full-length recombinant histone H4 using full-length recombinant SET8 in presence or absence of activated full-length recombinant PARP1.
    Figure Legend Snippet: (A) Detection of unbound 100 bp DNA ladder by TBE ethidium bromide-stained gel in supernatants (left lane) on GST-SET8 domains or mutant (M) using GST-pulldown assays (top, left side). Asterisks are representing shift of DNA on GST-SET8 157-352 amino acid protein or GST-SET8 full-length protein (FL) beads. Ponceau stain represents the amount of GST beads constructs used for the GST-pulldown (bottom, left side). (B) Different concentrations of recombinant full-length SET8 protein binding to DNA using EMSA to determine the equilibrium dissociation constant (Kd). (C) Detection of unbound mononucleosome by TBE ethidium bromide-stained gel in supernatants on GST beads versus GST-SET8 domains or mutant (M) beads using GST-pulldown assays. (D) Detection of unbound DNA (left side) or unbound mononucleosome (right side) by TBE ethidium bromide-stained gel in supernatants (top) on GST beads versus GST-SET8 FL beads using GST-pulldown assays. GST or GST-SET8 FL beads were poly ADP-ribosylated or not (with or without NAD) using full-length recombinant PARP1 as demonstrated by western blot using anti-ADP ribose antibody (middle). Ponceau stain gel (bottom) represents the amount of GST bead constructs used for the GST-pulldown and the ADP-ribosylation western blot analysis. (E) SET8 histone methyltransferase assay on full-length recombinant histone H4 using full-length recombinant SET8 in presence or absence of activated full-length recombinant PARP1.

    Techniques Used: Staining, Mutagenesis, Construct, Recombinant, Protein Binding, Western Blot, HMT Assay

    h4  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95

    Structured Review

    New England Biolabs h4
    H4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/h4/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    h4 - by Bioz Stars, 2023-01
    95/100 stars

    Images

    human recombinant histone 4  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95

    Structured Review

    New England Biolabs human recombinant histone 4
    Human Recombinant Histone 4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human recombinant histone 4/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human recombinant histone 4 - by Bioz Stars, 2023-01
    95/100 stars

    Images

    histone h4 custom  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95

    Structured Review

    New England Biolabs histone h4 custom
    (A) Representative immunofluorescence images of dHL60 cells showing extracellular citrullinated histone <t>H4</t> staining after 4 hours <t>of</t> <t>ionomycin</t> (iono) treatment with staining for H4Cit (green) and nuclei (blue). Scale bars, 100 μm. (B) Quantification of extracellular histone H4 staining in dHL60 cells after ionomycin treatment. Mean ± SD is shown ( n = 3 independent experiments). (C) Representative immunofluorescence images of human neutrophils showing extracellular citrullinated histone H4 staining after 4 hours of ionomycin treatment with staining for H4Cit (green) and nuclei (blue). Scale bars, 100 μm. (D) Quantification of extracellular histone H4 staining in human neutrophils after ionomycin treatment. Mean ± SD is shown ( n = 3 independent experiments). **, P < 0.01. Ctrl, unstimulated control.
    Histone H4 Custom, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h4 custom/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    histone h4 custom - by Bioz Stars, 2023-01
    95/100 stars

    Images

    1) Product Images from "Neutrophil stimulation with citrullinated histone H4 slows down calcium influx and reduces NET formation compared with native histone H4"

    Article Title: Neutrophil stimulation with citrullinated histone H4 slows down calcium influx and reduces NET formation compared with native histone H4

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0251726

    (A) Representative immunofluorescence images of dHL60 cells showing extracellular citrullinated histone H4 staining after 4 hours of ionomycin (iono) treatment with staining for H4Cit (green) and nuclei (blue). Scale bars, 100 μm. (B) Quantification of extracellular histone H4 staining in dHL60 cells after ionomycin treatment. Mean ± SD is shown ( n = 3 independent experiments). (C) Representative immunofluorescence images of human neutrophils showing extracellular citrullinated histone H4 staining after 4 hours of ionomycin treatment with staining for H4Cit (green) and nuclei (blue). Scale bars, 100 μm. (D) Quantification of extracellular histone H4 staining in human neutrophils after ionomycin treatment. Mean ± SD is shown ( n = 3 independent experiments). **, P < 0.01. Ctrl, unstimulated control.
    Figure Legend Snippet: (A) Representative immunofluorescence images of dHL60 cells showing extracellular citrullinated histone H4 staining after 4 hours of ionomycin (iono) treatment with staining for H4Cit (green) and nuclei (blue). Scale bars, 100 μm. (B) Quantification of extracellular histone H4 staining in dHL60 cells after ionomycin treatment. Mean ± SD is shown ( n = 3 independent experiments). (C) Representative immunofluorescence images of human neutrophils showing extracellular citrullinated histone H4 staining after 4 hours of ionomycin treatment with staining for H4Cit (green) and nuclei (blue). Scale bars, 100 μm. (D) Quantification of extracellular histone H4 staining in human neutrophils after ionomycin treatment. Mean ± SD is shown ( n = 3 independent experiments). **, P < 0.01. Ctrl, unstimulated control.

    Techniques Used: Immunofluorescence, Staining

    (A) Immunofluorescence images revealed reduced histone H4-induced NET formation with pretreatment by PAD inhibitor Cl-amidine or intracellular calcium chelator BAPTA, AM in dHL60 cells. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (B) Quantification of NET formation in dHL60 cells after the different stimuli in (A). Mean ± SD is shown ( n = 3 independent experiments). (C) Immunofluorescence images revealed reduced histone H4-induced NET formation with pretreatment by PAD inhibitor Cl-amidine or intracellular calcium chelator BAPTA, AM in human neutrophils. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (D) Quantification of NET formation in human neutrophils after the different stimuli in (C). Mean ± SD is shown ( n = 3 independent experiments). (E) Immunofluorescence images revealed reduced histone H4-induced NET formation with pretreatment of intracellular calcium chelator BAPTA, AM in mouse neutrophils. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (F) Quantification of histone H4-induced NET formation in mouse neutrophils with or without pretreatment of calcium chelator BAPTA, AM. Mean ± SD is shown ( n = 3 or 4 mice). (G) Immunofluorescence images of wild type and Padi4 knockout mouse neutrophils after histone H4 treatment. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (H) Quantification of histone H4-induced NET formation in wild type and Padi4 knockout mouse neutrophils. Mean ± SD is shown ( n = 5 for wild type mice and 3 for Padi4 knockout mice). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. Ctrl, unstimulated control.
    Figure Legend Snippet: (A) Immunofluorescence images revealed reduced histone H4-induced NET formation with pretreatment by PAD inhibitor Cl-amidine or intracellular calcium chelator BAPTA, AM in dHL60 cells. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (B) Quantification of NET formation in dHL60 cells after the different stimuli in (A). Mean ± SD is shown ( n = 3 independent experiments). (C) Immunofluorescence images revealed reduced histone H4-induced NET formation with pretreatment by PAD inhibitor Cl-amidine or intracellular calcium chelator BAPTA, AM in human neutrophils. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (D) Quantification of NET formation in human neutrophils after the different stimuli in (C). Mean ± SD is shown ( n = 3 independent experiments). (E) Immunofluorescence images revealed reduced histone H4-induced NET formation with pretreatment of intracellular calcium chelator BAPTA, AM in mouse neutrophils. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (F) Quantification of histone H4-induced NET formation in mouse neutrophils with or without pretreatment of calcium chelator BAPTA, AM. Mean ± SD is shown ( n = 3 or 4 mice). (G) Immunofluorescence images of wild type and Padi4 knockout mouse neutrophils after histone H4 treatment. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (H) Quantification of histone H4-induced NET formation in wild type and Padi4 knockout mouse neutrophils. Mean ± SD is shown ( n = 5 for wild type mice and 3 for Padi4 knockout mice). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. Ctrl, unstimulated control.

    Techniques Used: Immunofluorescence, Knock-Out

    (A) Immunofluorescence images of NET formation in dHL60 cells after treatments with native or citrullinated histone H4 or PAD4 alone as control. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (B) Quantification of NET formation in dHL60 cells after treatments with native or citrullinated histone H4 or PAD4 alone as control. Mean ± SD is shown ( n = 3 for controls and 5 for histone H4 treatments). (C) PAD4, H3Cit, and H4Cit protein levels were determined by western blot analysis in dHL60 cells after treatments with native or citrullinated histone H4 or PAD4 alone as control. The results are representative of three experiments. (D) Immunofluorescence images of NET formation in human neutrophils after treatment with native or citrullinated histone H4. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (E) Quantification of NET formation in human neutrophils after treatment with native or citrullinated histone H4. Mean ± SD is shown ( n = 4 independent experiments). (F) PAD4 and H3Cit protein levels were determined by western blot analysis in human neutrophils after treatment with native or citrullinated histone H4. The results are representative of three experiments. (G) Immunofluorescence images of NET formation in mouse neutrophils after treatment with native or citrullinated histone H4. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (H) Quantification of NET formation in mouse neutrophils after treatment with native or citrullinated histone H4. Mean ± SD is shown ( n = 5 different fields from 3 independent experiments). *, P < 0.05; **, P < 0.01; ns, not significant. Ctrl, unstimulated control.
    Figure Legend Snippet: (A) Immunofluorescence images of NET formation in dHL60 cells after treatments with native or citrullinated histone H4 or PAD4 alone as control. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (B) Quantification of NET formation in dHL60 cells after treatments with native or citrullinated histone H4 or PAD4 alone as control. Mean ± SD is shown ( n = 3 for controls and 5 for histone H4 treatments). (C) PAD4, H3Cit, and H4Cit protein levels were determined by western blot analysis in dHL60 cells after treatments with native or citrullinated histone H4 or PAD4 alone as control. The results are representative of three experiments. (D) Immunofluorescence images of NET formation in human neutrophils after treatment with native or citrullinated histone H4. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (E) Quantification of NET formation in human neutrophils after treatment with native or citrullinated histone H4. Mean ± SD is shown ( n = 4 independent experiments). (F) PAD4 and H3Cit protein levels were determined by western blot analysis in human neutrophils after treatment with native or citrullinated histone H4. The results are representative of three experiments. (G) Immunofluorescence images of NET formation in mouse neutrophils after treatment with native or citrullinated histone H4. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (H) Quantification of NET formation in mouse neutrophils after treatment with native or citrullinated histone H4. Mean ± SD is shown ( n = 5 different fields from 3 independent experiments). *, P < 0.05; **, P < 0.01; ns, not significant. Ctrl, unstimulated control.

    Techniques Used: Immunofluorescence, Western Blot

    (A) Fluo-4 kinetic fluorescent readings of dHL60 cells in resting condition (Starting point) and during 2 h of above indicated treatments (Stimulants). (B) Quantification of area under the curve in (A). The area under the curve from histone H4 or ionomycin treatment was normalized to control treatments. Mean ± SD is shown ( n = 4 independent experiments). (C) Quantification of time to reach half of the maximal signal from native and citrullinated histone H4 treatments in (A). Mean ± SD is shown ( n = 4 independent experiments). (D) Fluo-4 kinetic fluorescent readings of human neutrophils in resting condition (Starting point) and during 2 h of above indicated treatments (Stimulants). (E) Quantification of area under the curve in (D). The area under the curve from histone H4 or ionomycin treatment was normalized to control treatments. Mean ± SD is shown ( n = 5 independent experiments). (F) Quantification of time to reach half of the maximal signal from native and citrullinated histone H4 treatments in (D). Mean ± SD is shown ( n = 5 independent experiments); *, P < 0.05, **, P < 0.01; ns, not significant. Ctrl, unstimulated control.
    Figure Legend Snippet: (A) Fluo-4 kinetic fluorescent readings of dHL60 cells in resting condition (Starting point) and during 2 h of above indicated treatments (Stimulants). (B) Quantification of area under the curve in (A). The area under the curve from histone H4 or ionomycin treatment was normalized to control treatments. Mean ± SD is shown ( n = 4 independent experiments). (C) Quantification of time to reach half of the maximal signal from native and citrullinated histone H4 treatments in (A). Mean ± SD is shown ( n = 4 independent experiments). (D) Fluo-4 kinetic fluorescent readings of human neutrophils in resting condition (Starting point) and during 2 h of above indicated treatments (Stimulants). (E) Quantification of area under the curve in (D). The area under the curve from histone H4 or ionomycin treatment was normalized to control treatments. Mean ± SD is shown ( n = 5 independent experiments). (F) Quantification of time to reach half of the maximal signal from native and citrullinated histone H4 treatments in (D). Mean ± SD is shown ( n = 5 independent experiments); *, P < 0.05, **, P < 0.01; ns, not significant. Ctrl, unstimulated control.

    Techniques Used:

    recombinant histone h4  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95

    Structured Review

    New England Biolabs recombinant histone h4
    (A) Representative confocal images of neutrophils treated with Phil82 strain of IAV. DNA was stained blue with DAPI, Phil82 IAV green with FITC, and <t>histone</t> <t>H4</t> red with Alexa Fluor 594. The image shown was taken at 100x magnification. (B) 96-well plates were coated with H4 or BSA and then incubated with Alexa-labeled Phil82, PR8 or Cal09 IAV strains for 45 minutes. Binding of H4 to IAVs was determined by detecting the fluorescence intensity. (C) Viral aggregation was measured by increased light transmission through stirred suspensions of Phil82 IAV. (D) Histone H4 was pre-incubated with FITC-labeled IAV for 30 minutes, and then it was added to the neutrophils for 45 minutes. Uptake of virus by neutrophils was measured by flow cytometry using Trypan blue to quench extracellular fluorescence. Control cells that were not treated with Phil-FITC are shown in each histogram overlay (gray lines). Cells treated only with Phil-FITC but not histone H4 are shown in each histogram overlay (black lines) Cells incubated with virus that was treated with H4 are shown as solid black peaks. (E) Fold of mean fluorescence intensity from flow cytometry is shown (PBS indicates virus alone incubated in PBS without H4). N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).
    Recombinant Histone H4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant histone h4/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant histone h4 - by Bioz Stars, 2023-01
    95/100 stars

    Images

    1) Product Images from "Histone H4 potentiates neutrophil inflammatory responses to influenza A virus: Down-modulation by H4 binding to C-reactive protein and Surfactant protein D"

    Article Title: Histone H4 potentiates neutrophil inflammatory responses to influenza A virus: Down-modulation by H4 binding to C-reactive protein and Surfactant protein D

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0247605

    (A) Representative confocal images of neutrophils treated with Phil82 strain of IAV. DNA was stained blue with DAPI, Phil82 IAV green with FITC, and histone H4 red with Alexa Fluor 594. The image shown was taken at 100x magnification. (B) 96-well plates were coated with H4 or BSA and then incubated with Alexa-labeled Phil82, PR8 or Cal09 IAV strains for 45 minutes. Binding of H4 to IAVs was determined by detecting the fluorescence intensity. (C) Viral aggregation was measured by increased light transmission through stirred suspensions of Phil82 IAV. (D) Histone H4 was pre-incubated with FITC-labeled IAV for 30 minutes, and then it was added to the neutrophils for 45 minutes. Uptake of virus by neutrophils was measured by flow cytometry using Trypan blue to quench extracellular fluorescence. Control cells that were not treated with Phil-FITC are shown in each histogram overlay (gray lines). Cells treated only with Phil-FITC but not histone H4 are shown in each histogram overlay (black lines) Cells incubated with virus that was treated with H4 are shown as solid black peaks. (E) Fold of mean fluorescence intensity from flow cytometry is shown (PBS indicates virus alone incubated in PBS without H4). N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).
    Figure Legend Snippet: (A) Representative confocal images of neutrophils treated with Phil82 strain of IAV. DNA was stained blue with DAPI, Phil82 IAV green with FITC, and histone H4 red with Alexa Fluor 594. The image shown was taken at 100x magnification. (B) 96-well plates were coated with H4 or BSA and then incubated with Alexa-labeled Phil82, PR8 or Cal09 IAV strains for 45 minutes. Binding of H4 to IAVs was determined by detecting the fluorescence intensity. (C) Viral aggregation was measured by increased light transmission through stirred suspensions of Phil82 IAV. (D) Histone H4 was pre-incubated with FITC-labeled IAV for 30 minutes, and then it was added to the neutrophils for 45 minutes. Uptake of virus by neutrophils was measured by flow cytometry using Trypan blue to quench extracellular fluorescence. Control cells that were not treated with Phil-FITC are shown in each histogram overlay (gray lines). Cells treated only with Phil-FITC but not histone H4 are shown in each histogram overlay (black lines) Cells incubated with virus that was treated with H4 are shown as solid black peaks. (E) Fold of mean fluorescence intensity from flow cytometry is shown (PBS indicates virus alone incubated in PBS without H4). N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).

    Techniques Used: Staining, Incubation, Labeling, Binding Assay, Fluorescence, Transmission Assay, Flow Cytometry

    Human neutrophils were treated with control buffer alone (PBS), H4, IAV or the combination of IAV and H4. Samples were made up either with PBS containing calcium (PBS++) (A and C) or PBS without calcium (PBS wo) (B and D). In panels C and D neutrophils were pre-incubated with 20 μM BAPTA-AM for 10 minutes before the experiments the chelate intracellular calcium. Neutrophils were treated with PBS, histone H4, Phil82 IAV strain or combinations of histone H4 and Phil82 strain. For the combinations, histone H4 and the virus were pre-incubated for 30 minutes prior to the experiment. Hydrogen peroxide production was measured by assessing the reduction in scopoletin fluorescence. Mean fluorescence curves are shown in the left panels, and fold changes of area over the curve (AOC) for those experiments are shown in the right panel. The MOI for these experiments was 40. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).
    Figure Legend Snippet: Human neutrophils were treated with control buffer alone (PBS), H4, IAV or the combination of IAV and H4. Samples were made up either with PBS containing calcium (PBS++) (A and C) or PBS without calcium (PBS wo) (B and D). In panels C and D neutrophils were pre-incubated with 20 μM BAPTA-AM for 10 minutes before the experiments the chelate intracellular calcium. Neutrophils were treated with PBS, histone H4, Phil82 IAV strain or combinations of histone H4 and Phil82 strain. For the combinations, histone H4 and the virus were pre-incubated for 30 minutes prior to the experiment. Hydrogen peroxide production was measured by assessing the reduction in scopoletin fluorescence. Mean fluorescence curves are shown in the left panels, and fold changes of area over the curve (AOC) for those experiments are shown in the right panel. The MOI for these experiments was 40. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).

    Techniques Used: Incubation, Fluorescence

    Intracellular free calcium changes in neutrophils treated with PBS, fMLP, histone H4, Phil82 IAV strain or combinations of histone H4 and Phil82 strain were measured by detecting the fluorescence of Fura-2 AM loaded neutrophils. For the combinations, histone H4 and the virus were pre-incubated for 30 minutes prior to the experiment. All samples were added to the cells at 135 seconds. Mean fluorescence curves are shown in panel A and fold changes of area under the curve (AUC) for these experiments are shown panel B. The MOI for these experiments was 40. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05).
    Figure Legend Snippet: Intracellular free calcium changes in neutrophils treated with PBS, fMLP, histone H4, Phil82 IAV strain or combinations of histone H4 and Phil82 strain were measured by detecting the fluorescence of Fura-2 AM loaded neutrophils. For the combinations, histone H4 and the virus were pre-incubated for 30 minutes prior to the experiment. All samples were added to the cells at 135 seconds. Mean fluorescence curves are shown in panel A and fold changes of area under the curve (AUC) for these experiments are shown panel B. The MOI for these experiments was 40. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05).

    Techniques Used: Fluorescence, Incubation

    96-well plates were coated with 10 μg/mL histone H4 overnight. In panel A different concentrations of CRP were added to the plate and the bound CRP was detected by anti-CRP antibody (HRP conjugated) and TMB substrate. Fold changes in light absorption are shown. (B) Different concentrations of SP-D were added to the H4 coated plates and the bound SP-D was detected by anti-SP-D antibody (HRP conjugated) and TMB substrate. Fold changes in light absorption are shown. (C) Different concentrations of NCRD were added to the plate and the bound NCRD was detected by anti-S antibody (HRP conjugated) and TMB substrate. Fold changes in light absorption are shown. (D) Different concentrations of D325A+R343V double mutant NCRD (*NCRD) were added to the H4 coated plates and the bond *NCRD was detected by anti-S antibody (HRP conjugated) and TMB substrate. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).
    Figure Legend Snippet: 96-well plates were coated with 10 μg/mL histone H4 overnight. In panel A different concentrations of CRP were added to the plate and the bound CRP was detected by anti-CRP antibody (HRP conjugated) and TMB substrate. Fold changes in light absorption are shown. (B) Different concentrations of SP-D were added to the H4 coated plates and the bound SP-D was detected by anti-SP-D antibody (HRP conjugated) and TMB substrate. Fold changes in light absorption are shown. (C) Different concentrations of NCRD were added to the plate and the bound NCRD was detected by anti-S antibody (HRP conjugated) and TMB substrate. Fold changes in light absorption are shown. (D) Different concentrations of D325A+R343V double mutant NCRD (*NCRD) were added to the H4 coated plates and the bond *NCRD was detected by anti-S antibody (HRP conjugated) and TMB substrate. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).

    Techniques Used: Mutagenesis

    (A) 2 μg or 4 μg of histone H4 was incubated with 2 μg of CRP for 30 minutes. After centrifugation, pellet and supernatant from each sample were run on a gel separately and stained with Gelcode blue. A representative result is shown. Similar effects were seen when H4 was pre-incubated with either SP-D or wild type NCRD (B). Densitometry readings were made on pellet bands from gels from 4 replicate experiments and these are shown in panel C for CRP, SP-D and NCRD bands and in panel D for histone bands. The effect of carrying out incubation of NCRD in solution containing EDTA or maltose (10 mM) is shown in panel E. Results of panels C-E are mean ± S.E.M of 4 experiments and in each case significantly more protein (p< 0.05) was found in the pellets when proteins were co-incubated than when single proteins were tested.
    Figure Legend Snippet: (A) 2 μg or 4 μg of histone H4 was incubated with 2 μg of CRP for 30 minutes. After centrifugation, pellet and supernatant from each sample were run on a gel separately and stained with Gelcode blue. A representative result is shown. Similar effects were seen when H4 was pre-incubated with either SP-D or wild type NCRD (B). Densitometry readings were made on pellet bands from gels from 4 replicate experiments and these are shown in panel C for CRP, SP-D and NCRD bands and in panel D for histone bands. The effect of carrying out incubation of NCRD in solution containing EDTA or maltose (10 mM) is shown in panel E. Results of panels C-E are mean ± S.E.M of 4 experiments and in each case significantly more protein (p< 0.05) was found in the pellets when proteins were co-incubated than when single proteins were tested.

    Techniques Used: Incubation, Centrifugation, Staining

    (A) Viral aggregation : 2 μg/mL of histone H4 or 4 μg/mL of CRP were incubated alone or together for 30 minutes before the experiments. Viral aggregation was measured by increased light absorption through stirred suspensions of Phil82 IAV. (B) Viral neutralization : 1.25 or 2.5 μg/mL of histone H4 or 8 μg/mL of CRP were incubated alone or together for 30 minutes before the experiments, as indicated. IAV neutralization assessed using the fluorescent focus assay for detection of viral nucleoprotein. Phil82 strain was used. (C, D) Neutrophil uptake of IAV : 20 or 40 μg/mL of histone H4 or 4 μg/mL of CRP were incubated alone or together for 30 minutes before the experiments. Each sample was pre-incubated with FITC-labeled Phi82 IAV for 30 minutes, and then this was added to the neutrophils for 45 minutes. Uptake of virus by neutrophils was measured by flow cytometry. Cells treated only with Phil-FITC are shown in each histogram overlay (black peaks). Fold of mean fluorescence intensity from flow cytometry is shown in B. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).
    Figure Legend Snippet: (A) Viral aggregation : 2 μg/mL of histone H4 or 4 μg/mL of CRP were incubated alone or together for 30 minutes before the experiments. Viral aggregation was measured by increased light absorption through stirred suspensions of Phil82 IAV. (B) Viral neutralization : 1.25 or 2.5 μg/mL of histone H4 or 8 μg/mL of CRP were incubated alone or together for 30 minutes before the experiments, as indicated. IAV neutralization assessed using the fluorescent focus assay for detection of viral nucleoprotein. Phil82 strain was used. (C, D) Neutrophil uptake of IAV : 20 or 40 μg/mL of histone H4 or 4 μg/mL of CRP were incubated alone or together for 30 minutes before the experiments. Each sample was pre-incubated with FITC-labeled Phi82 IAV for 30 minutes, and then this was added to the neutrophils for 45 minutes. Uptake of virus by neutrophils was measured by flow cytometry. Cells treated only with Phil-FITC are shown in each histogram overlay (black peaks). Fold of mean fluorescence intensity from flow cytometry is shown in B. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).

    Techniques Used: Incubation, Neutralization, Labeling, Flow Cytometry, Fluorescence

    Hydrogen peroxide production in neutrophils on exposure to PBS++ control buffer, fMLP, or 40 μg/mL histone H4 pre-incubated with or without 80 μg/mL CRP, 8 μg/mL D325A+R343V double mutant NCRD (*NCRD) or 16 μg/mL SP-D was measured by assessing the reduction in scopoletin fluorescence. Panel A shows mean scopoletin fluorescence and Panel B show fold changes of area over the curve (AOC).
    Figure Legend Snippet: Hydrogen peroxide production in neutrophils on exposure to PBS++ control buffer, fMLP, or 40 μg/mL histone H4 pre-incubated with or without 80 μg/mL CRP, 8 μg/mL D325A+R343V double mutant NCRD (*NCRD) or 16 μg/mL SP-D was measured by assessing the reduction in scopoletin fluorescence. Panel A shows mean scopoletin fluorescence and Panel B show fold changes of area over the curve (AOC).

    Techniques Used: Incubation, Mutagenesis, Fluorescence

    Mean fluorescence curves are shown in the left panels, and fold changes of area under the curve (AUC) for these experiments are shown on the right. Intracellular free calcium was detected by loading neutrophils with Fura-2 AM. (A) Intracellular free calcium changes in neutrophils on exposure to PBS++ control buffer, fMLP, 40 μg/mL histone H4, 80 μg/mL CRP or the combination of histone H4 and CRP. (B) Intracellular free calcium changes in neutrophils on exposure to PBS++ control buffer, fMLP, 40 μg/mL histone H4, 16 μg/mL SP-D or the combination of histone H4 and SP-D. (C) Intracellular free calcium changes in neutrophils on exposure to PBS++ control buffer, fMLP, 40 μg/mL histone H4, 8 μg/mL D325A+R343V double mutant NCRD (*NCRD) or the combination of histone H4 and mutant.
    Figure Legend Snippet: Mean fluorescence curves are shown in the left panels, and fold changes of area under the curve (AUC) for these experiments are shown on the right. Intracellular free calcium was detected by loading neutrophils with Fura-2 AM. (A) Intracellular free calcium changes in neutrophils on exposure to PBS++ control buffer, fMLP, 40 μg/mL histone H4, 80 μg/mL CRP or the combination of histone H4 and CRP. (B) Intracellular free calcium changes in neutrophils on exposure to PBS++ control buffer, fMLP, 40 μg/mL histone H4, 16 μg/mL SP-D or the combination of histone H4 and SP-D. (C) Intracellular free calcium changes in neutrophils on exposure to PBS++ control buffer, fMLP, 40 μg/mL histone H4, 8 μg/mL D325A+R343V double mutant NCRD (*NCRD) or the combination of histone H4 and mutant.

    Techniques Used: Fluorescence, Mutagenesis

    Neutrophils were pre-incubated with 50 mM Di-OC5 5 minutes followed by addition of 40 μg/ml of H4 at time zero. Histone H4 alone or histone H4 which had been pre-incubated with CRP was added to neutrophils and depolarization of the neutrophil membrane was indicated by reduction in fluorescence. Results are mean ± SEM for 4 experiments with p< 0.05 when comparing H4 alone to H4 plus CRP (ANOVA).
    Figure Legend Snippet: Neutrophils were pre-incubated with 50 mM Di-OC5 5 minutes followed by addition of 40 μg/ml of H4 at time zero. Histone H4 alone or histone H4 which had been pre-incubated with CRP was added to neutrophils and depolarization of the neutrophil membrane was indicated by reduction in fluorescence. Results are mean ± SEM for 4 experiments with p< 0.05 when comparing H4 alone to H4 plus CRP (ANOVA).

    Techniques Used: Incubation, Fluorescence

    Neutrophils were treated with PBS++ control buffer, 40 μg/mL histone H4 alone or histone that had been pre-incubated with either CRP, SP-D, or 8 μg/mL D325A+R343V double mutant NCRD (*NCRD). In addition, CRP, SP-D or *NCRD were added alone. Panel A shows effects of the treatments on MPO release into the cell supernatant and panel B shows effects of the treatments on caspase 3 activity as measured with the Z-DEVD–AMC substrate. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).
    Figure Legend Snippet: Neutrophils were treated with PBS++ control buffer, 40 μg/mL histone H4 alone or histone that had been pre-incubated with either CRP, SP-D, or 8 μg/mL D325A+R343V double mutant NCRD (*NCRD). In addition, CRP, SP-D or *NCRD were added alone. Panel A shows effects of the treatments on MPO release into the cell supernatant and panel B shows effects of the treatments on caspase 3 activity as measured with the Z-DEVD–AMC substrate. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).

    Techniques Used: Incubation, Mutagenesis, Activity Assay

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95
    New England Biolabs human histone h4
    Human Histone H4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human histone h4/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human histone h4 - by Bioz Stars, 2023-01
    95/100 stars
      Buy from Supplier

    95
    New England Biolabs recombinant human histone h4
    A. Crosslinking <t>of</t> <t>H3–H4</t> . Histones H3 and H4 were incubated for the indicated minutes and then crosslinked with formaldehyde before separation on a 15% SDS/PAGE. The blot was probed separately with anti-H3 and anti-H4. B. Effect of Asf1 and TLK1B on H3–H4 dimer and tetramer formation. Reactions containing the indicated combinations of H3, H4, Asf1, and TLK1B (all in equivalent amount) were crosslinked as described in Methods and immunoblotted for H4 or H3. C. Western blots for Asf1 and TLK1B. The indicated reactions as in panel B were run in duplicate lanes and immunoblotted for Asf1, H3, or TLK1B (the gel was run for a longer time than in panel B to separate the larger proteins). For antibody controls, lane 1 contained only Asf1, and lane 7 contained only TLK1B. The positions of the cross-linked complexes identified by mobility and immunoreactivity are indicated. D. TLK1B and Asf1 bind each other stoichiometrically. Reactions contained 5 μg of GST-TLK1B and varying amounts of Asf1, as indicated. After 10 min at room temperature, the samples were adsorbed on GSH-Sepharose and analyzed for bound and unbound fractions after separation on a 10% SDS/PAGE, which was stained with Coomassie blue. E. Interaction of TLK1B and Asf1 and the effect of ATP. GST-TLK1B and Asf1 (1 μg each) were incubated for 10 min at room temperature with and without 10 or 100 μM ATP, before analysis by GSH-Sepharose pull-down. The 10% SDS/PAGE gel was stained with Coomassie blue. F. MNase digestion of pBluescript assembled into pseudo-nucleosomes. In the left panel, naked supercoiled plasmid was digested with MNase for the indicated time. In the right panel, the plasmid was first incubated with equimolar H3 and H4 in high salt and then step-dialyzed as described in , before MNase digestion. The DNA was re-extracted from the reactions with Geneclean and run on a 1.5% agarose/TAE gel. The resulting ~120 bp ladder (a bit shorter than the repetitive 146 bp of nucleosomal DNA) is indicative of formation of a chromatinized template. The positions of the bands of a 100-bp ladder (GenRuler, Fermentas) are indicated.
    Recombinant Human Histone H4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant human histone h4/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant human histone h4 - by Bioz Stars, 2023-01
    95/100 stars
      Buy from Supplier

    95
    New England Biolabs m2504s
    KEY RESOURCES TABLE
    M2504s, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m2504s/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    m2504s - by Bioz Stars, 2023-01
    95/100 stars
      Buy from Supplier

    95
    New England Biolabs h4 histone
    KEY RESOURCES TABLE
    H4 Histone, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/h4 histone/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    h4 histone - by Bioz Stars, 2023-01
    95/100 stars
      Buy from Supplier

    95
    New England Biolabs h4
    KEY RESOURCES TABLE
    H4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/h4/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    h4 - by Bioz Stars, 2023-01
    95/100 stars
      Buy from Supplier

    95
    New England Biolabs human recombinant histone 4
    KEY RESOURCES TABLE
    Human Recombinant Histone 4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human recombinant histone 4/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human recombinant histone 4 - by Bioz Stars, 2023-01
    95/100 stars
      Buy from Supplier

    95
    New England Biolabs histone h4 custom
    (A) Representative immunofluorescence images of dHL60 cells showing extracellular citrullinated histone <t>H4</t> staining after 4 hours <t>of</t> <t>ionomycin</t> (iono) treatment with staining for H4Cit (green) and nuclei (blue). Scale bars, 100 μm. (B) Quantification of extracellular histone H4 staining in dHL60 cells after ionomycin treatment. Mean ± SD is shown ( n = 3 independent experiments). (C) Representative immunofluorescence images of human neutrophils showing extracellular citrullinated histone H4 staining after 4 hours of ionomycin treatment with staining for H4Cit (green) and nuclei (blue). Scale bars, 100 μm. (D) Quantification of extracellular histone H4 staining in human neutrophils after ionomycin treatment. Mean ± SD is shown ( n = 3 independent experiments). **, P < 0.01. Ctrl, unstimulated control.
    Histone H4 Custom, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h4 custom/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    histone h4 custom - by Bioz Stars, 2023-01
    95/100 stars
      Buy from Supplier

    95
    New England Biolabs recombinant histone h4
    (A) Representative confocal images of neutrophils treated with Phil82 strain of IAV. DNA was stained blue with DAPI, Phil82 IAV green with FITC, and <t>histone</t> <t>H4</t> red with Alexa Fluor 594. The image shown was taken at 100x magnification. (B) 96-well plates were coated with H4 or BSA and then incubated with Alexa-labeled Phil82, PR8 or Cal09 IAV strains for 45 minutes. Binding of H4 to IAVs was determined by detecting the fluorescence intensity. (C) Viral aggregation was measured by increased light transmission through stirred suspensions of Phil82 IAV. (D) Histone H4 was pre-incubated with FITC-labeled IAV for 30 minutes, and then it was added to the neutrophils for 45 minutes. Uptake of virus by neutrophils was measured by flow cytometry using Trypan blue to quench extracellular fluorescence. Control cells that were not treated with Phil-FITC are shown in each histogram overlay (gray lines). Cells treated only with Phil-FITC but not histone H4 are shown in each histogram overlay (black lines) Cells incubated with virus that was treated with H4 are shown as solid black peaks. (E) Fold of mean fluorescence intensity from flow cytometry is shown (PBS indicates virus alone incubated in PBS without H4). N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).
    Recombinant Histone H4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant histone h4/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant histone h4 - by Bioz Stars, 2023-01
    95/100 stars
      Buy from Supplier

    Image Search Results


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

    Journal: BMC Research Notes

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

    doi: 10.1186/1756-0500-2-128

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

    Article Snippet: Recombinant human histone H4 was purchased from NE Biolabs and bovine H3 from Roche.

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

    KEY RESOURCES TABLE

    Journal: Cell reports

    Article Title: Induction of broadly reactive influenza antibodies increases susceptibility to autoimmunity

    doi: 10.1016/j.celrep.2022.110482

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: H4 (histone) , New England BioLabs , Cat# M2504S.

    Techniques: Recombinant, Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Stripping Membranes, Microarray, Software, Imaging

    KEY RESOURCES TABLE

    Journal: Cell reports

    Article Title: Induction of broadly reactive influenza antibodies increases susceptibility to autoimmunity

    doi: 10.1016/j.celrep.2022.110482

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: H4 (histone) , New England BioLabs , Cat# M2504S.

    Techniques: Recombinant, Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Stripping Membranes, Microarray, Software, Imaging

    (A) Representative immunofluorescence images of dHL60 cells showing extracellular citrullinated histone H4 staining after 4 hours of ionomycin (iono) treatment with staining for H4Cit (green) and nuclei (blue). Scale bars, 100 μm. (B) Quantification of extracellular histone H4 staining in dHL60 cells after ionomycin treatment. Mean ± SD is shown ( n = 3 independent experiments). (C) Representative immunofluorescence images of human neutrophils showing extracellular citrullinated histone H4 staining after 4 hours of ionomycin treatment with staining for H4Cit (green) and nuclei (blue). Scale bars, 100 μm. (D) Quantification of extracellular histone H4 staining in human neutrophils after ionomycin treatment. Mean ± SD is shown ( n = 3 independent experiments). **, P < 0.01. Ctrl, unstimulated control.

    Journal: PLoS ONE

    Article Title: Neutrophil stimulation with citrullinated histone H4 slows down calcium influx and reduces NET formation compared with native histone H4

    doi: 10.1371/journal.pone.0251726

    Figure Lengend Snippet: (A) Representative immunofluorescence images of dHL60 cells showing extracellular citrullinated histone H4 staining after 4 hours of ionomycin (iono) treatment with staining for H4Cit (green) and nuclei (blue). Scale bars, 100 μm. (B) Quantification of extracellular histone H4 staining in dHL60 cells after ionomycin treatment. Mean ± SD is shown ( n = 3 independent experiments). (C) Representative immunofluorescence images of human neutrophils showing extracellular citrullinated histone H4 staining after 4 hours of ionomycin treatment with staining for H4Cit (green) and nuclei (blue). Scale bars, 100 μm. (D) Quantification of extracellular histone H4 staining in human neutrophils after ionomycin treatment. Mean ± SD is shown ( n = 3 independent experiments). **, P < 0.01. Ctrl, unstimulated control.

    Article Snippet: For NETs induction, cells were simulated with 4 μmol/L calcium ionophore ionomycin (Invitrogen), 20 ng/μL native (NEB) or citrullinated histone H4 (custom), or 4 ng/μL recombinant human PAD4 (custom) control for 4 h at 37°C and 5% CO2 in phenol red-free RPMI 1640 medium supplemented with 10 mmol/L HEPES.

    Techniques: Immunofluorescence, Staining

    (A) Immunofluorescence images revealed reduced histone H4-induced NET formation with pretreatment by PAD inhibitor Cl-amidine or intracellular calcium chelator BAPTA, AM in dHL60 cells. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (B) Quantification of NET formation in dHL60 cells after the different stimuli in (A). Mean ± SD is shown ( n = 3 independent experiments). (C) Immunofluorescence images revealed reduced histone H4-induced NET formation with pretreatment by PAD inhibitor Cl-amidine or intracellular calcium chelator BAPTA, AM in human neutrophils. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (D) Quantification of NET formation in human neutrophils after the different stimuli in (C). Mean ± SD is shown ( n = 3 independent experiments). (E) Immunofluorescence images revealed reduced histone H4-induced NET formation with pretreatment of intracellular calcium chelator BAPTA, AM in mouse neutrophils. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (F) Quantification of histone H4-induced NET formation in mouse neutrophils with or without pretreatment of calcium chelator BAPTA, AM. Mean ± SD is shown ( n = 3 or 4 mice). (G) Immunofluorescence images of wild type and Padi4 knockout mouse neutrophils after histone H4 treatment. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (H) Quantification of histone H4-induced NET formation in wild type and Padi4 knockout mouse neutrophils. Mean ± SD is shown ( n = 5 for wild type mice and 3 for Padi4 knockout mice). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. Ctrl, unstimulated control.

    Journal: PLoS ONE

    Article Title: Neutrophil stimulation with citrullinated histone H4 slows down calcium influx and reduces NET formation compared with native histone H4

    doi: 10.1371/journal.pone.0251726

    Figure Lengend Snippet: (A) Immunofluorescence images revealed reduced histone H4-induced NET formation with pretreatment by PAD inhibitor Cl-amidine or intracellular calcium chelator BAPTA, AM in dHL60 cells. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (B) Quantification of NET formation in dHL60 cells after the different stimuli in (A). Mean ± SD is shown ( n = 3 independent experiments). (C) Immunofluorescence images revealed reduced histone H4-induced NET formation with pretreatment by PAD inhibitor Cl-amidine or intracellular calcium chelator BAPTA, AM in human neutrophils. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (D) Quantification of NET formation in human neutrophils after the different stimuli in (C). Mean ± SD is shown ( n = 3 independent experiments). (E) Immunofluorescence images revealed reduced histone H4-induced NET formation with pretreatment of intracellular calcium chelator BAPTA, AM in mouse neutrophils. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (F) Quantification of histone H4-induced NET formation in mouse neutrophils with or without pretreatment of calcium chelator BAPTA, AM. Mean ± SD is shown ( n = 3 or 4 mice). (G) Immunofluorescence images of wild type and Padi4 knockout mouse neutrophils after histone H4 treatment. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (H) Quantification of histone H4-induced NET formation in wild type and Padi4 knockout mouse neutrophils. Mean ± SD is shown ( n = 5 for wild type mice and 3 for Padi4 knockout mice). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. Ctrl, unstimulated control.

    Article Snippet: For NETs induction, cells were simulated with 4 μmol/L calcium ionophore ionomycin (Invitrogen), 20 ng/μL native (NEB) or citrullinated histone H4 (custom), or 4 ng/μL recombinant human PAD4 (custom) control for 4 h at 37°C and 5% CO2 in phenol red-free RPMI 1640 medium supplemented with 10 mmol/L HEPES.

    Techniques: Immunofluorescence, Knock-Out

    (A) Immunofluorescence images of NET formation in dHL60 cells after treatments with native or citrullinated histone H4 or PAD4 alone as control. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (B) Quantification of NET formation in dHL60 cells after treatments with native or citrullinated histone H4 or PAD4 alone as control. Mean ± SD is shown ( n = 3 for controls and 5 for histone H4 treatments). (C) PAD4, H3Cit, and H4Cit protein levels were determined by western blot analysis in dHL60 cells after treatments with native or citrullinated histone H4 or PAD4 alone as control. The results are representative of three experiments. (D) Immunofluorescence images of NET formation in human neutrophils after treatment with native or citrullinated histone H4. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (E) Quantification of NET formation in human neutrophils after treatment with native or citrullinated histone H4. Mean ± SD is shown ( n = 4 independent experiments). (F) PAD4 and H3Cit protein levels were determined by western blot analysis in human neutrophils after treatment with native or citrullinated histone H4. The results are representative of three experiments. (G) Immunofluorescence images of NET formation in mouse neutrophils after treatment with native or citrullinated histone H4. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (H) Quantification of NET formation in mouse neutrophils after treatment with native or citrullinated histone H4. Mean ± SD is shown ( n = 5 different fields from 3 independent experiments). *, P < 0.05; **, P < 0.01; ns, not significant. Ctrl, unstimulated control.

    Journal: PLoS ONE

    Article Title: Neutrophil stimulation with citrullinated histone H4 slows down calcium influx and reduces NET formation compared with native histone H4

    doi: 10.1371/journal.pone.0251726

    Figure Lengend Snippet: (A) Immunofluorescence images of NET formation in dHL60 cells after treatments with native or citrullinated histone H4 or PAD4 alone as control. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (B) Quantification of NET formation in dHL60 cells after treatments with native or citrullinated histone H4 or PAD4 alone as control. Mean ± SD is shown ( n = 3 for controls and 5 for histone H4 treatments). (C) PAD4, H3Cit, and H4Cit protein levels were determined by western blot analysis in dHL60 cells after treatments with native or citrullinated histone H4 or PAD4 alone as control. The results are representative of three experiments. (D) Immunofluorescence images of NET formation in human neutrophils after treatment with native or citrullinated histone H4. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (E) Quantification of NET formation in human neutrophils after treatment with native or citrullinated histone H4. Mean ± SD is shown ( n = 4 independent experiments). (F) PAD4 and H3Cit protein levels were determined by western blot analysis in human neutrophils after treatment with native or citrullinated histone H4. The results are representative of three experiments. (G) Immunofluorescence images of NET formation in mouse neutrophils after treatment with native or citrullinated histone H4. H3Cit (green) and nuclei (blue). Scale bars, 100 μm. (H) Quantification of NET formation in mouse neutrophils after treatment with native or citrullinated histone H4. Mean ± SD is shown ( n = 5 different fields from 3 independent experiments). *, P < 0.05; **, P < 0.01; ns, not significant. Ctrl, unstimulated control.

    Article Snippet: For NETs induction, cells were simulated with 4 μmol/L calcium ionophore ionomycin (Invitrogen), 20 ng/μL native (NEB) or citrullinated histone H4 (custom), or 4 ng/μL recombinant human PAD4 (custom) control for 4 h at 37°C and 5% CO2 in phenol red-free RPMI 1640 medium supplemented with 10 mmol/L HEPES.

    Techniques: Immunofluorescence, Western Blot

    (A) Fluo-4 kinetic fluorescent readings of dHL60 cells in resting condition (Starting point) and during 2 h of above indicated treatments (Stimulants). (B) Quantification of area under the curve in (A). The area under the curve from histone H4 or ionomycin treatment was normalized to control treatments. Mean ± SD is shown ( n = 4 independent experiments). (C) Quantification of time to reach half of the maximal signal from native and citrullinated histone H4 treatments in (A). Mean ± SD is shown ( n = 4 independent experiments). (D) Fluo-4 kinetic fluorescent readings of human neutrophils in resting condition (Starting point) and during 2 h of above indicated treatments (Stimulants). (E) Quantification of area under the curve in (D). The area under the curve from histone H4 or ionomycin treatment was normalized to control treatments. Mean ± SD is shown ( n = 5 independent experiments). (F) Quantification of time to reach half of the maximal signal from native and citrullinated histone H4 treatments in (D). Mean ± SD is shown ( n = 5 independent experiments); *, P < 0.05, **, P < 0.01; ns, not significant. Ctrl, unstimulated control.

    Journal: PLoS ONE

    Article Title: Neutrophil stimulation with citrullinated histone H4 slows down calcium influx and reduces NET formation compared with native histone H4

    doi: 10.1371/journal.pone.0251726

    Figure Lengend Snippet: (A) Fluo-4 kinetic fluorescent readings of dHL60 cells in resting condition (Starting point) and during 2 h of above indicated treatments (Stimulants). (B) Quantification of area under the curve in (A). The area under the curve from histone H4 or ionomycin treatment was normalized to control treatments. Mean ± SD is shown ( n = 4 independent experiments). (C) Quantification of time to reach half of the maximal signal from native and citrullinated histone H4 treatments in (A). Mean ± SD is shown ( n = 4 independent experiments). (D) Fluo-4 kinetic fluorescent readings of human neutrophils in resting condition (Starting point) and during 2 h of above indicated treatments (Stimulants). (E) Quantification of area under the curve in (D). The area under the curve from histone H4 or ionomycin treatment was normalized to control treatments. Mean ± SD is shown ( n = 5 independent experiments). (F) Quantification of time to reach half of the maximal signal from native and citrullinated histone H4 treatments in (D). Mean ± SD is shown ( n = 5 independent experiments); *, P < 0.05, **, P < 0.01; ns, not significant. Ctrl, unstimulated control.

    Article Snippet: For NETs induction, cells were simulated with 4 μmol/L calcium ionophore ionomycin (Invitrogen), 20 ng/μL native (NEB) or citrullinated histone H4 (custom), or 4 ng/μL recombinant human PAD4 (custom) control for 4 h at 37°C and 5% CO2 in phenol red-free RPMI 1640 medium supplemented with 10 mmol/L HEPES.

    Techniques:

    (A) Representative confocal images of neutrophils treated with Phil82 strain of IAV. DNA was stained blue with DAPI, Phil82 IAV green with FITC, and histone H4 red with Alexa Fluor 594. The image shown was taken at 100x magnification. (B) 96-well plates were coated with H4 or BSA and then incubated with Alexa-labeled Phil82, PR8 or Cal09 IAV strains for 45 minutes. Binding of H4 to IAVs was determined by detecting the fluorescence intensity. (C) Viral aggregation was measured by increased light transmission through stirred suspensions of Phil82 IAV. (D) Histone H4 was pre-incubated with FITC-labeled IAV for 30 minutes, and then it was added to the neutrophils for 45 minutes. Uptake of virus by neutrophils was measured by flow cytometry using Trypan blue to quench extracellular fluorescence. Control cells that were not treated with Phil-FITC are shown in each histogram overlay (gray lines). Cells treated only with Phil-FITC but not histone H4 are shown in each histogram overlay (black lines) Cells incubated with virus that was treated with H4 are shown as solid black peaks. (E) Fold of mean fluorescence intensity from flow cytometry is shown (PBS indicates virus alone incubated in PBS without H4). N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).

    Journal: PLoS ONE

    Article Title: Histone H4 potentiates neutrophil inflammatory responses to influenza A virus: Down-modulation by H4 binding to C-reactive protein and Surfactant protein D

    doi: 10.1371/journal.pone.0247605

    Figure Lengend Snippet: (A) Representative confocal images of neutrophils treated with Phil82 strain of IAV. DNA was stained blue with DAPI, Phil82 IAV green with FITC, and histone H4 red with Alexa Fluor 594. The image shown was taken at 100x magnification. (B) 96-well plates were coated with H4 or BSA and then incubated with Alexa-labeled Phil82, PR8 or Cal09 IAV strains for 45 minutes. Binding of H4 to IAVs was determined by detecting the fluorescence intensity. (C) Viral aggregation was measured by increased light transmission through stirred suspensions of Phil82 IAV. (D) Histone H4 was pre-incubated with FITC-labeled IAV for 30 minutes, and then it was added to the neutrophils for 45 minutes. Uptake of virus by neutrophils was measured by flow cytometry using Trypan blue to quench extracellular fluorescence. Control cells that were not treated with Phil-FITC are shown in each histogram overlay (gray lines). Cells treated only with Phil-FITC but not histone H4 are shown in each histogram overlay (black lines) Cells incubated with virus that was treated with H4 are shown as solid black peaks. (E) Fold of mean fluorescence intensity from flow cytometry is shown (PBS indicates virus alone incubated in PBS without H4). N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).

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

    Techniques: Staining, Incubation, Labeling, Binding Assay, Fluorescence, Transmission Assay, Flow Cytometry

    Human neutrophils were treated with control buffer alone (PBS), H4, IAV or the combination of IAV and H4. Samples were made up either with PBS containing calcium (PBS++) (A and C) or PBS without calcium (PBS wo) (B and D). In panels C and D neutrophils were pre-incubated with 20 μM BAPTA-AM for 10 minutes before the experiments the chelate intracellular calcium. Neutrophils were treated with PBS, histone H4, Phil82 IAV strain or combinations of histone H4 and Phil82 strain. For the combinations, histone H4 and the virus were pre-incubated for 30 minutes prior to the experiment. Hydrogen peroxide production was measured by assessing the reduction in scopoletin fluorescence. Mean fluorescence curves are shown in the left panels, and fold changes of area over the curve (AOC) for those experiments are shown in the right panel. The MOI for these experiments was 40. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).

    Journal: PLoS ONE

    Article Title: Histone H4 potentiates neutrophil inflammatory responses to influenza A virus: Down-modulation by H4 binding to C-reactive protein and Surfactant protein D

    doi: 10.1371/journal.pone.0247605

    Figure Lengend Snippet: Human neutrophils were treated with control buffer alone (PBS), H4, IAV or the combination of IAV and H4. Samples were made up either with PBS containing calcium (PBS++) (A and C) or PBS without calcium (PBS wo) (B and D). In panels C and D neutrophils were pre-incubated with 20 μM BAPTA-AM for 10 minutes before the experiments the chelate intracellular calcium. Neutrophils were treated with PBS, histone H4, Phil82 IAV strain or combinations of histone H4 and Phil82 strain. For the combinations, histone H4 and the virus were pre-incubated for 30 minutes prior to the experiment. Hydrogen peroxide production was measured by assessing the reduction in scopoletin fluorescence. Mean fluorescence curves are shown in the left panels, and fold changes of area over the curve (AOC) for those experiments are shown in the right panel. The MOI for these experiments was 40. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).

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

    Techniques: Incubation, Fluorescence

    Intracellular free calcium changes in neutrophils treated with PBS, fMLP, histone H4, Phil82 IAV strain or combinations of histone H4 and Phil82 strain were measured by detecting the fluorescence of Fura-2 AM loaded neutrophils. For the combinations, histone H4 and the virus were pre-incubated for 30 minutes prior to the experiment. All samples were added to the cells at 135 seconds. Mean fluorescence curves are shown in panel A and fold changes of area under the curve (AUC) for these experiments are shown panel B. The MOI for these experiments was 40. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05).

    Journal: PLoS ONE

    Article Title: Histone H4 potentiates neutrophil inflammatory responses to influenza A virus: Down-modulation by H4 binding to C-reactive protein and Surfactant protein D

    doi: 10.1371/journal.pone.0247605

    Figure Lengend Snippet: Intracellular free calcium changes in neutrophils treated with PBS, fMLP, histone H4, Phil82 IAV strain or combinations of histone H4 and Phil82 strain were measured by detecting the fluorescence of Fura-2 AM loaded neutrophils. For the combinations, histone H4 and the virus were pre-incubated for 30 minutes prior to the experiment. All samples were added to the cells at 135 seconds. Mean fluorescence curves are shown in panel A and fold changes of area under the curve (AUC) for these experiments are shown panel B. The MOI for these experiments was 40. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05).

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

    Techniques: Fluorescence, Incubation

    96-well plates were coated with 10 μg/mL histone H4 overnight. In panel A different concentrations of CRP were added to the plate and the bound CRP was detected by anti-CRP antibody (HRP conjugated) and TMB substrate. Fold changes in light absorption are shown. (B) Different concentrations of SP-D were added to the H4 coated plates and the bound SP-D was detected by anti-SP-D antibody (HRP conjugated) and TMB substrate. Fold changes in light absorption are shown. (C) Different concentrations of NCRD were added to the plate and the bound NCRD was detected by anti-S antibody (HRP conjugated) and TMB substrate. Fold changes in light absorption are shown. (D) Different concentrations of D325A+R343V double mutant NCRD (*NCRD) were added to the H4 coated plates and the bond *NCRD was detected by anti-S antibody (HRP conjugated) and TMB substrate. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).

    Journal: PLoS ONE

    Article Title: Histone H4 potentiates neutrophil inflammatory responses to influenza A virus: Down-modulation by H4 binding to C-reactive protein and Surfactant protein D

    doi: 10.1371/journal.pone.0247605

    Figure Lengend Snippet: 96-well plates were coated with 10 μg/mL histone H4 overnight. In panel A different concentrations of CRP were added to the plate and the bound CRP was detected by anti-CRP antibody (HRP conjugated) and TMB substrate. Fold changes in light absorption are shown. (B) Different concentrations of SP-D were added to the H4 coated plates and the bound SP-D was detected by anti-SP-D antibody (HRP conjugated) and TMB substrate. Fold changes in light absorption are shown. (C) Different concentrations of NCRD were added to the plate and the bound NCRD was detected by anti-S antibody (HRP conjugated) and TMB substrate. Fold changes in light absorption are shown. (D) Different concentrations of D325A+R343V double mutant NCRD (*NCRD) were added to the H4 coated plates and the bond *NCRD was detected by anti-S antibody (HRP conjugated) and TMB substrate. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).

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

    Techniques: Mutagenesis

    (A) 2 μg or 4 μg of histone H4 was incubated with 2 μg of CRP for 30 minutes. After centrifugation, pellet and supernatant from each sample were run on a gel separately and stained with Gelcode blue. A representative result is shown. Similar effects were seen when H4 was pre-incubated with either SP-D or wild type NCRD (B). Densitometry readings were made on pellet bands from gels from 4 replicate experiments and these are shown in panel C for CRP, SP-D and NCRD bands and in panel D for histone bands. The effect of carrying out incubation of NCRD in solution containing EDTA or maltose (10 mM) is shown in panel E. Results of panels C-E are mean ± S.E.M of 4 experiments and in each case significantly more protein (p< 0.05) was found in the pellets when proteins were co-incubated than when single proteins were tested.

    Journal: PLoS ONE

    Article Title: Histone H4 potentiates neutrophil inflammatory responses to influenza A virus: Down-modulation by H4 binding to C-reactive protein and Surfactant protein D

    doi: 10.1371/journal.pone.0247605

    Figure Lengend Snippet: (A) 2 μg or 4 μg of histone H4 was incubated with 2 μg of CRP for 30 minutes. After centrifugation, pellet and supernatant from each sample were run on a gel separately and stained with Gelcode blue. A representative result is shown. Similar effects were seen when H4 was pre-incubated with either SP-D or wild type NCRD (B). Densitometry readings were made on pellet bands from gels from 4 replicate experiments and these are shown in panel C for CRP, SP-D and NCRD bands and in panel D for histone bands. The effect of carrying out incubation of NCRD in solution containing EDTA or maltose (10 mM) is shown in panel E. Results of panels C-E are mean ± S.E.M of 4 experiments and in each case significantly more protein (p< 0.05) was found in the pellets when proteins were co-incubated than when single proteins were tested.

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

    Techniques: Incubation, Centrifugation, Staining

    (A) Viral aggregation : 2 μg/mL of histone H4 or 4 μg/mL of CRP were incubated alone or together for 30 minutes before the experiments. Viral aggregation was measured by increased light absorption through stirred suspensions of Phil82 IAV. (B) Viral neutralization : 1.25 or 2.5 μg/mL of histone H4 or 8 μg/mL of CRP were incubated alone or together for 30 minutes before the experiments, as indicated. IAV neutralization assessed using the fluorescent focus assay for detection of viral nucleoprotein. Phil82 strain was used. (C, D) Neutrophil uptake of IAV : 20 or 40 μg/mL of histone H4 or 4 μg/mL of CRP were incubated alone or together for 30 minutes before the experiments. Each sample was pre-incubated with FITC-labeled Phi82 IAV for 30 minutes, and then this was added to the neutrophils for 45 minutes. Uptake of virus by neutrophils was measured by flow cytometry. Cells treated only with Phil-FITC are shown in each histogram overlay (black peaks). Fold of mean fluorescence intensity from flow cytometry is shown in B. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).

    Journal: PLoS ONE

    Article Title: Histone H4 potentiates neutrophil inflammatory responses to influenza A virus: Down-modulation by H4 binding to C-reactive protein and Surfactant protein D

    doi: 10.1371/journal.pone.0247605

    Figure Lengend Snippet: (A) Viral aggregation : 2 μg/mL of histone H4 or 4 μg/mL of CRP were incubated alone or together for 30 minutes before the experiments. Viral aggregation was measured by increased light absorption through stirred suspensions of Phil82 IAV. (B) Viral neutralization : 1.25 or 2.5 μg/mL of histone H4 or 8 μg/mL of CRP were incubated alone or together for 30 minutes before the experiments, as indicated. IAV neutralization assessed using the fluorescent focus assay for detection of viral nucleoprotein. Phil82 strain was used. (C, D) Neutrophil uptake of IAV : 20 or 40 μg/mL of histone H4 or 4 μg/mL of CRP were incubated alone or together for 30 minutes before the experiments. Each sample was pre-incubated with FITC-labeled Phi82 IAV for 30 minutes, and then this was added to the neutrophils for 45 minutes. Uptake of virus by neutrophils was measured by flow cytometry. Cells treated only with Phil-FITC are shown in each histogram overlay (black peaks). Fold of mean fluorescence intensity from flow cytometry is shown in B. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).

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

    Techniques: Incubation, Neutralization, Labeling, Flow Cytometry, Fluorescence

    Hydrogen peroxide production in neutrophils on exposure to PBS++ control buffer, fMLP, or 40 μg/mL histone H4 pre-incubated with or without 80 μg/mL CRP, 8 μg/mL D325A+R343V double mutant NCRD (*NCRD) or 16 μg/mL SP-D was measured by assessing the reduction in scopoletin fluorescence. Panel A shows mean scopoletin fluorescence and Panel B show fold changes of area over the curve (AOC).

    Journal: PLoS ONE

    Article Title: Histone H4 potentiates neutrophil inflammatory responses to influenza A virus: Down-modulation by H4 binding to C-reactive protein and Surfactant protein D

    doi: 10.1371/journal.pone.0247605

    Figure Lengend Snippet: Hydrogen peroxide production in neutrophils on exposure to PBS++ control buffer, fMLP, or 40 μg/mL histone H4 pre-incubated with or without 80 μg/mL CRP, 8 μg/mL D325A+R343V double mutant NCRD (*NCRD) or 16 μg/mL SP-D was measured by assessing the reduction in scopoletin fluorescence. Panel A shows mean scopoletin fluorescence and Panel B show fold changes of area over the curve (AOC).

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

    Techniques: Incubation, Mutagenesis, Fluorescence

    Mean fluorescence curves are shown in the left panels, and fold changes of area under the curve (AUC) for these experiments are shown on the right. Intracellular free calcium was detected by loading neutrophils with Fura-2 AM. (A) Intracellular free calcium changes in neutrophils on exposure to PBS++ control buffer, fMLP, 40 μg/mL histone H4, 80 μg/mL CRP or the combination of histone H4 and CRP. (B) Intracellular free calcium changes in neutrophils on exposure to PBS++ control buffer, fMLP, 40 μg/mL histone H4, 16 μg/mL SP-D or the combination of histone H4 and SP-D. (C) Intracellular free calcium changes in neutrophils on exposure to PBS++ control buffer, fMLP, 40 μg/mL histone H4, 8 μg/mL D325A+R343V double mutant NCRD (*NCRD) or the combination of histone H4 and mutant.

    Journal: PLoS ONE

    Article Title: Histone H4 potentiates neutrophil inflammatory responses to influenza A virus: Down-modulation by H4 binding to C-reactive protein and Surfactant protein D

    doi: 10.1371/journal.pone.0247605

    Figure Lengend Snippet: Mean fluorescence curves are shown in the left panels, and fold changes of area under the curve (AUC) for these experiments are shown on the right. Intracellular free calcium was detected by loading neutrophils with Fura-2 AM. (A) Intracellular free calcium changes in neutrophils on exposure to PBS++ control buffer, fMLP, 40 μg/mL histone H4, 80 μg/mL CRP or the combination of histone H4 and CRP. (B) Intracellular free calcium changes in neutrophils on exposure to PBS++ control buffer, fMLP, 40 μg/mL histone H4, 16 μg/mL SP-D or the combination of histone H4 and SP-D. (C) Intracellular free calcium changes in neutrophils on exposure to PBS++ control buffer, fMLP, 40 μg/mL histone H4, 8 μg/mL D325A+R343V double mutant NCRD (*NCRD) or the combination of histone H4 and mutant.

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

    Techniques: Fluorescence, Mutagenesis

    Neutrophils were pre-incubated with 50 mM Di-OC5 5 minutes followed by addition of 40 μg/ml of H4 at time zero. Histone H4 alone or histone H4 which had been pre-incubated with CRP was added to neutrophils and depolarization of the neutrophil membrane was indicated by reduction in fluorescence. Results are mean ± SEM for 4 experiments with p< 0.05 when comparing H4 alone to H4 plus CRP (ANOVA).

    Journal: PLoS ONE

    Article Title: Histone H4 potentiates neutrophil inflammatory responses to influenza A virus: Down-modulation by H4 binding to C-reactive protein and Surfactant protein D

    doi: 10.1371/journal.pone.0247605

    Figure Lengend Snippet: Neutrophils were pre-incubated with 50 mM Di-OC5 5 minutes followed by addition of 40 μg/ml of H4 at time zero. Histone H4 alone or histone H4 which had been pre-incubated with CRP was added to neutrophils and depolarization of the neutrophil membrane was indicated by reduction in fluorescence. Results are mean ± SEM for 4 experiments with p< 0.05 when comparing H4 alone to H4 plus CRP (ANOVA).

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

    Techniques: Incubation, Fluorescence

    Neutrophils were treated with PBS++ control buffer, 40 μg/mL histone H4 alone or histone that had been pre-incubated with either CRP, SP-D, or 8 μg/mL D325A+R343V double mutant NCRD (*NCRD). In addition, CRP, SP-D or *NCRD were added alone. Panel A shows effects of the treatments on MPO release into the cell supernatant and panel B shows effects of the treatments on caspase 3 activity as measured with the Z-DEVD–AMC substrate. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).

    Journal: PLoS ONE

    Article Title: Histone H4 potentiates neutrophil inflammatory responses to influenza A virus: Down-modulation by H4 binding to C-reactive protein and Surfactant protein D

    doi: 10.1371/journal.pone.0247605

    Figure Lengend Snippet: Neutrophils were treated with PBS++ control buffer, 40 μg/mL histone H4 alone or histone that had been pre-incubated with either CRP, SP-D, or 8 μg/mL D325A+R343V double mutant NCRD (*NCRD). In addition, CRP, SP-D or *NCRD were added alone. Panel A shows effects of the treatments on MPO release into the cell supernatant and panel B shows effects of the treatments on caspase 3 activity as measured with the Z-DEVD–AMC substrate. N = 5. Results are presented as mean ± S.E.M (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).

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

    Techniques: Incubation, Mutagenesis, Activity Assay