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: H2B (histone) , New England BioLabs , Cat# M2505S.

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: Briefly, microtiter plates (Nunc) were coated with 0.15 μg/well of histones H2B, H1, H3 or H4 (New England BioLabs) in PBS.

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

Journal: Communications Biology

Article Title: COMMD4 functions with the histone H2A-H2B dimer for the timely repair of DNA double-strand breaks

doi: 10.1038/s42003-021-01998-2

Figure Lengend Snippet: a Empty vector (FLAG) and COMMD4-FLAG were immunoprecipitated from HeLa cells and the co-eluting proteins were immunoblotted with antibodies shown. b Immunoprecipitation as a , but this time cells were treated with 6 Gy IR. c A direct interaction for COMMD4 and H2B ± recombinant ATM. d A direct interaction between COMMD4 and H2B ± recombinant ATM and the ATM inhibitor (ATMi) KU-55933. e FLAG and FLAG-tagged H2B WT, and S14E mutant were immunoprecipitated from HeLa cells and the co-eluting proteins were immunoblotted with antibodies against COMMD4 and H2B. IgG heavy chain shows the loading. f Direct interaction between COMMD4 and H2B demonstrating the specific binding region within H2B. No binding was seen in the negative control with only the beads and recombinant COMMD4 (beads), while expression of His-tagged COMMD4 (COMMD4, right most lane) is seen. Schematic on the right shows peptide regions within H2B and Supplementary Table shows the peptide sequences. g Direct interaction between RNF40 and H2B demonstrating the specific binding regions within H2B. Expression of recombinant RNF40 is seen in the control lane. h A three dimensional structure (PDB code 2RVQ) of the heterodimer H2A-H2B wherein H2A and H2B are shown in blue and red ribbon representation, respectively. Tails of histones are disordered and in extended conformation. The phosphorylation sites are shown as spheres.

Article Snippet: In total, 300 ng of recombinant COMMD4, purified from the pET-28a expression vector, was incubated with either 300 ng of H2A (NEB) or H2B (NEB) for 1 h at 4 °C in NP40 buffer.

Techniques: Plasmid Preparation, Immunoprecipitation, Recombinant, Mutagenesis, Binding Assay, Negative Control, Expressing

Journal: Communications Biology

Article Title: COMMD4 functions with the histone H2A-H2B dimer for the timely repair of DNA double-strand breaks

doi: 10.1038/s42003-021-01998-2

Figure Lengend Snippet: a Immunoblot showing H2B monoubiquitination in control and COMMD4-depleted cells ±5 μg/ml of Actinomycin D and ±irradiation. b Immunofluorescence showing RNF40 protein levels with 6 Gy IR at 0 and 4 h post-IR treatment in control and COMMD4-depleted cells. Scale bar denotes 5 μm. Fifty cells were quantified per condition. c In vitro ubiquitination assay of H2B ± recombinant COMMD4 or with COMMD4 and without the E2 enzyme. Monoubiquitination and polyubiquitination of H2B is shown. d Direct interaction between COMMD4 and H2A/H2B, demonstrating that COMMD4 preferentially binds H2B. e In vitro direct interaction between H2A, H2B and COMMD4 ± recombinant CK2. H2B was used pull-out the complexes and shows loading. f Same as e , however, COMMD4 was used to pull-out the complexes. g Direct interaction between H2A, H2B and COMMD4, in the absence and presence of purified nucleosomes, ±recombinant ATM and CK2. COMMD4 was used pull-out the complexes and shows the loading.

Article Snippet: In total, 300 ng of recombinant COMMD4, purified from the pET-28a expression vector, was incubated with either 300 ng of H2A (NEB) or H2B (NEB) for 1 h at 4 °C in NP40 buffer.

Techniques: Western Blot, Irradiation, Immunofluorescence, In Vitro, Ubiquitin Assay, Recombinant, Purification