Journal: The Journal of Biological Chemistry

Article Title: Multivalent nucleosome scaffolding by bromodomain and extraterminal domain tandem bromodomains

doi: 10.1016/j.jbc.2025.108289

Figure Lengend Snippet: BRD4 tandem bromodomains scaffold acetylated nucleosomes in vitro in AlphaScreen assays. A , AlphaScreen assay schematic. B , AlphaScreen of WT tandem BRD4 ( dark blue ), N140F ( dark gray ), or N433F ( light gray ) tandem bromodomains binding to a recombinant H4K5ac nucleosome (n = 3), where error bars represent SD. C , AlphaScreen of WT tandem BRD2 ( black ), BRD3 ( dark gray ), or BRDT ( light gray ) tandem bromodomains binding to a recombinant H4K5ac nucleosome (n = 3), where error bars represent SD. BRD2, bromodomain-containing protein 2; BRD3, bromodomain-containing protein 3; BRD4, bromodomain-containing protein 4; BRDT, bromodomain-containing protein, testis-associated.

Article Snippet: Biotinylated recombinant H4K5ac nucleosome (EpiCypher, 16–0352) was diluted to 1.5 μM in nucleosome storage buffer (10 mM Tris–HCl, 25 mM NaCl, 1 mM EDTA, 2 mM DTT, pH 7.5), then diluted in salt-free nucleosome assay buffer (20 mM Hepes, 0.01% v/v NP-40 alternative, 0.01% w/v BSA, 1 mM DTT, pH 7.5) to create an 8 × (80 nM) nucleosome stock.

Techniques: In Vitro, Amplified Luminescent Proximity Homogenous Assay, Binding Assay, Recombinant

Journal: Genomics, Proteomics & Bioinformatics

Article Title: Diversified Application of Barcoded PLATO (PLATO-BC) Platform for Identification of Protein Interactions

doi: 10.1016/j.gpb.2018.12.010

Figure Lengend Snippet: Identification of protein binders for BETi JQ1 by using PLATO-BC assay A. Rank of ORF enrichment from the PLATO-BC assays for biotinylated JQ1 (Bio-JQ1) relative to free biotin (Bio). 12,492 ORFs were ranked according to the ratio of Bio-JQ1/Bio (from the smallest to the largest). The red line shows the cut-off value. A known protein target of JQ1, BRD2, was ranked as a top hit (red dot). B. Hit validation of JQ1 protein targets identified from the PLATO-BC assay. pET-DEST42-EWSR1/EYA3/RBM14/BD1/BD2 was transformed into the E. coli DE3 strain. V5-His6-tagged EWSR1/EYA3/RBM14/BD1/BD2 proteins were purified and subjected to protein pull-down assays for Bio-JQ1 or Bio. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody. C. Interaction of EWSR1 with H4ac peptide in vitro . V5-His6-tagged EWSR1 protein was purified and subjected to protein pull-down assays for biotinylated, acetylated or non-acetylated H4 (Bio-H4ac or Bio-H4) peptide. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody. D. Interaction of EWSR1 with H4ac in cells. pcDNA-DEST40-EWSR1 was transiently transfected in HEK293T cells. Cell lysate was prepared and subjected to the protein immunoprecipitation assays for V5-His6-tagged EWSR1 using an anti-V5 mouse antibody or a mouse IgG (mIgG) control. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-H4ac antibody. E. JQ1 interrupts the interaction of EWSR1 with H4ac peptide in vitro . V5-His6-tagged EWSR1 protein was purified and incubated with JQ1 (at 1 μM or 10 μM) or DMSO, prior to the protein pull-down assays for Bio-H4ac or free biotin (Bio). Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody or HRP-conjugated streptavidin to detect EWSR1 or H4ac respectively. BETi, bromodomain and extraterminal domain inhibitor; BRD2, bromodomain containing 2; H4ac, acetylated histone H4; EWSR1, Ewing sarcoma breakpoint region 1; EYA3, EYA transcriptional coactivator and phosphatase 3; RBM14, RNA-binding protein 14; BD1, bromodomain 1; BD2, bromodomain 2.

Article Snippet: Biotinylated histone H4 peptides (Catalog No. 120029) and biotinylated acetylated histone H4 peptides (Catalog No. 120047) were purchased from EpiCypher, Durham, NC.

Techniques: Biomarker Discovery, Transformation Assay, Purification, SDS Page, Western Blot, In Vitro, Transfection, Immunoprecipitation, Control, Incubation, RNA Binding Assay

Journal: Comparative Cytogenetics

Article Title: The epigenetic regulation of centromeres and telomeres in plants and animals

doi: 10.3897/CompCytogen.v14i2.51895

Figure Lengend Snippet: Epigenetic modifications of centromeric regions and their functions in plants and animals.

Article Snippet: Histone H4 acetylation (H4K5ac and H4K12ac) was also detected in Gallus (Brisson, 1760) cells as a modification necessary for CENP-A deposition ( ).

Techniques: Variant Assay, Activity Assay, Methylation