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

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

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

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

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

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

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

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

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

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

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

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

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