Journal: bioRxiv
Article Title: PBRM1-Dependent PBAF Targeting is Required for EMT and Metastasis in Breast Cancer
doi: 10.1101/2025.10.19.683137
Figure Lengend Snippet: (A) Coomassie gel of the purified recombinant proteins BD2, BD3, BD4, BD5 and the tandem BD2-5 used for peptide and nucleosome binding assays. (B) Schematic representation of EpiCypher’s Captify™ assay. (C) Table of EC 50 values (nM) of the different BDs for the indicated peptides obtained using the ALPHA/dCypher assay. HP1 binding to H3K9me3 peptide is used as a positive control in this assay. (D) Binding curves of tandem BD2-5 with the indicated peptides obtained using the ALPHA/dCypher assay. EC 50 (nM) values are indicated next to the corresponding curves. (E) Table of the relative EC 50 values (nM) of the different BDs for nucleosomes bearing the indicated histone modifications obtained using the ALPHA/dCypher assay. HP1 binding to H3K9me3 nucleosomes is used as a positive control in this assay. (F) Binding curves of tandem BD2-5 for nucleosomes bearing the indicated peptides obtained using the ALPHA/dCypher assay. HP1 binding to H3K9me3 peptide is used as a positive control in this assay. EC 50 values (nM) obtained for positive binders are indicated in the legend. (G and H) Metagene plots and heatmaps of ChIP-seq enrichment of Phf10, H3K14ac, H3K18ac, and H3K27ac at Phf10 binding sites in untreated (H) and 48h TGFβ1-treated (I) sgCt cells. (I) Correlation matrix with r-values between Phf10 and H3K14ac, H3K18ac, H3K27ac, and H3K4me3 ChIP-seq enrichment in untreated and 48h TGFβ1-treated sgCt cells. (J) Metagene plots and heatmaps of ChIP-seq enrichment of Phf10, H3K14ac, H3K18ac, and H3K27ac in untreated and 48h TGFβ1-treated sgCt cells. The top heatmap is at Phf10 binding sites in untreated cells and the bottom is Phf10 binding sites only found in TGFβ1-treated cells. (K and L) Genomic tracks of ChIP-seq enrichment of Phf10, H3K14ac, H3K18ac, and H3K27ac in untreated (M) and 48h-TGFβ1 treated (M) sgCt cells at constitutive locus Cpne2 and an inducible locus Tnfsf13b .
Article Snippet: Constructs encoding codon-optimized ORFs for bacterial expression of human PBRM1 BD2 (addgene #39013), BD3 (addgene #39030), BD4 (addgene #39103), BD5 (addgene #38999) and tandem BD2-5 (SGC construct ID #PB1A-c080) were transformed in BL21(DE3) for BDs, and BL21 Rosetta2 (DE3) pLysS for tandem BD2-5.
Techniques: Purification, Recombinant, Binding Assay, Positive Control, ChIP-sequencing
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