bd4  (Addgene inc)

Journal: Applied and Environmental Microbiology

Article Title: Genome evolution of Acinetobacter baylyi ADP1 during laboratory domestication: acquired mutations impact competence and metabolism

doi: 10.1128/aem.00936-25

Figure Lengend Snippet: A. baylyi strains form cell chains in minimal media. ( A ) Domestication in BD413 rev and ADP1(D) prevented the formation of cell chains in S2 medium with glucose. ( B ) Cell chains formed for all strains when succinate was used as a carbon source in S2 medium. ( C ) All strains, including BD4 rev , grew unicellularly in LB medium. The image of BD413 rev in LB is taken from the same field of view as in . Scale bars are 5 µM.

Article Snippet: ATCC 33304 , BD4 , ATCC , This study.

Techniques:

Journal: Applied and Environmental Microbiology

Article Title: Genome evolution of Acinetobacter baylyi ADP1 during laboratory domestication: acquired mutations impact competence and metabolism

doi: 10.1128/aem.00936-25

Figure Lengend Snippet: Transformation efficiency varies between domesticated A. baylyi strains. ( A ) Transformation frequencies of BD4 rev (black), BD413 rev (blue), ADP1(D) (orange), and ISx (green) strains incubated overnight with genomic DNA carrying a specR resistance gene. ( B ) Transformation frequencies of the same strains during log (squares) and stationary (triangles) phases. At each time point, each strain was incubated with transforming DNA for 30 min, then the remaining free DNA was digested by DNase I before plating to count transformants. ( C ) Transformation frequencies during log (squares) and stationary (triangles) phases for ADP1 and ISx compared to an ISx mutant with a truncated csrA gene (yellow). * P < 0.05 for Welch’s t -test.

Article Snippet: ATCC 33304 , BD4 , ATCC , This study.

Techniques: Transformation Assay, Incubation, Mutagenesis

Journal: Applied and Environmental Microbiology

Article Title: Genome evolution of Acinetobacter baylyi ADP1 during laboratory domestication: acquired mutations impact competence and metabolism

doi: 10.1128/aem.00936-25

Figure Lengend Snippet: Domestication impacted growth and catabolite repression in A. baylyi . Growth curves of A. baylyi strains in ( A ) LB medium and in S2 media with ( B ) 25 mM succinate, ( C ) 3 mM glucose, ( D ) 3 mM glucose and 2.5 mM succinate, ( E ) 2 mM benzoate, or ( F ) 20 mM vanillate. Lines represent the average OD 600 of six replicates each and error bars depict standard error. The strains tested were BD4 rev (black), BD413 rev (blue), ADP1(D) (orange), and ISx (green). E and F include an ISx mutant with the csrA truncation from ADP1(D) (yellow).

Article Snippet: ATCC 33304 , BD4 , ATCC , This study.

Techniques: Mutagenesis

Journal: Applied and Environmental Microbiology

Article Title: Genome evolution of Acinetobacter baylyi ADP1 during laboratory domestication: acquired mutations impact competence and metabolism

doi: 10.1128/aem.00936-25

Figure Lengend Snippet: ( A ) Mutations evolved in BD4 rev (MRV001) during 1 month of evolution in ABMS medium. Full details are provided in . ( B ) Potential post-transcriptional regulatory network inferred in A. baylyi from interactions between CsrA, Hfq, and RNase D. (1) Hfq protects csrA mRNA from degradation by RNase E . (2) CsrA proteins are bound by a CsrB-like small RNA . (3) CsrA binds to Shine-Dalgarno sites, preventing ribosome assembly on mRNAs . (4) CsrA protects mRNAs from degradation by RNase D ( rnd ). (5) CsrA protects small regulatory RNAs from degradation by RNases . Panel B created with Biorender.

Article Snippet: ATCC 33304 , BD4 , ATCC , This study.

Techniques:

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

Journal: The Journal of Biological Chemistry

Article Title: Cancer-associated polybromo-1 bromodomain 4 missense variants variably impact bromodomain ligand binding and cell growth suppression

doi: 10.1016/j.jbc.2024.107146

Figure Lengend Snippet: PBRM1 incurs frequent missense mutations in the context of cancer. A , proportion of cancer-associated PBRM1 variants by mutation type, as annotated in the COSMIC (Catalog of Somatic Mutations in Cancer) database. B , percentage of cancer-associated missense mutations of PBRM1 by functional domain, as annotated in the COSMIC database. C , cancer-associated missense mutations of PBRM1-BD4 per residue across the entire peptide sequence, as annotated in the COSMIC database. Bromodomains (BD; blue ), bromo-adjacent homology (BAH; red ), and high-mobility group (HMG; green ) domains are denoted, with domain boundaries determined from Pfam annotations ± 15 aa residues. D , structure-based sequence alignment of PBRM1 bromodomains, with the position of the four bromodomain ⍺-helices ( blue ) shown above. BD4 and the residues studied herein are highlighted within the sequence alignment. The heat map demonstrates the conservation level per residue across the six PBRM1 bromodomains, where higher conservation is indicated by reds and lower conservation is indicated by blues . E , Rosetta flexible peptide docking of an H3K14ac peptide ( gray ) from PBRM1-BD2 (PDB ID 2KTB ) to PBRM1-BD4 (PDB ID 3TLP ) ; mutated residues are represented as spheres and color-coded by the number of unique missense variants per residue examined in this study. H3K14ac, lysine-14 acetylation on histone H3; PBRM1, polybromo-1.

Article Snippet: Sections of the coding region for PBRM1-containing BD4 mutations were synthesized (Biomatik) and inserted into the digested TetO-Fuw-PBRM1 WT plasmid (Addgene plasmid #85746) ( ) using the In-FusionHD cloning kit (Clontech Laboratories, Inc) and confirmed by WideSeq (Purdue University).

Techniques: Mutagenesis, Functional Assay, Residue, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Cancer-associated polybromo-1 bromodomain 4 missense variants variably impact bromodomain ligand binding and cell growth suppression

doi: 10.1016/j.jbc.2024.107146

Figure Lengend Snippet: Cancer-associated PBRM1-BD4 missense variants primarily exhibit decreased protein stability with intact secondary and tertiary structure integrity. A , PBRM1-BD4 missense variant T m determined by SYPRO Orange thermal shift assay (controls shown in light gray , cancer-associated PBRM1-BD4 missense variants in dark gray ); ΔT m of PBRM1-BD4 variants compared to WT is also demonstrated (negative ΔT m denoted in blue , positive Δ T m in red ), where error bars represent SD; n = 9 for PBRM1-BD4 WT and all missense variants except N601K, where n = 6. B , heat map indicates a change in Gibbs free energy (ΔΔ G ) of PBRM1-BD4 missense variants compared to WT estimated by Rosetta modeling software (more divergent values shown in blue , less divergent in red ). C , correlation of SYPRO Orange thermal shift assay and ΔΔ G datasets, where horizontal error bars represent SD of protein melting temperatures determined by the SYPRO Orange thermal shift assay. D , CD spectrum of PBRM1-BD4 missense variant R540T. E , CD spectrum of PBRM1-BD4 missense variant R576P. F , 1 H-NMR spectra of PBRM1-BD4 WT and cancer-associated PBRM1-BD4 missense variants. The gray highlighted regions correspond to the spectral regions (backbone amide proton ∼6.5–9.5 ppm; saturated alkane methyl proton ∼0–1.25 ppm) used to assess variant tertiary structural integrity ( , ). BD4, fourth bromodomain; CD, circular dichroism; 1 H-NMR, one-dimensional proton NMR spectroscopy; PBRM1, polybromo-1; T m , protein melting temperature.

Article Snippet: Sections of the coding region for PBRM1-containing BD4 mutations were synthesized (Biomatik) and inserted into the digested TetO-Fuw-PBRM1 WT plasmid (Addgene plasmid #85746) ( ) using the In-FusionHD cloning kit (Clontech Laboratories, Inc) and confirmed by WideSeq (Purdue University).

Techniques: Variant Assay, Thermal Shift Assay, Software, Circular Dichroism, Proton NMR, Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Cancer-associated polybromo-1 bromodomain 4 missense variants variably impact bromodomain ligand binding and cell growth suppression

doi: 10.1016/j.jbc.2024.107146

Figure Lengend Snippet: Cancer-associated PBRM1-BD4 missense variants exhibit variable ligand binding capacity. A , heat map indicates PBRM1-BD4 variant (0.1–10 μM) binding of H3K14ac peptide (50 nM) compared to WT in AlphaScreen assays (n = 3). B , AlphaScreen titrations of PBRM1-BD4 P556S, M586I, and R576L (0.001–2 μM) against a biotinylated histone H3K14ac (50 nM) peptide (n = 3), where error bars represent SEM. C , EMSA of PBRM1-BD4 WT (0–50 μM) binding to 150 nM Widom 601 DNA (representative of n = 2). D , EMSA of PBRM1-BD4 variants (20 μM) binding to 150 nM Widom 601 DNA (representative of n = 2). Alpha, amplified luminescent proximity homogeneous assay; BD4, fourth bromodomain; EMSA, electrophoretic mobility shift assay; H3K14ac, lysine-14 acetylation on histone H3; PBRM1, polybromo-1.

Article Snippet: Sections of the coding region for PBRM1-containing BD4 mutations were synthesized (Biomatik) and inserted into the digested TetO-Fuw-PBRM1 WT plasmid (Addgene plasmid #85746) ( ) using the In-FusionHD cloning kit (Clontech Laboratories, Inc) and confirmed by WideSeq (Purdue University).

Techniques: Ligand Binding Assay, Variant Assay, Binding Assay, Amplified Luminescent Proximity Homogenous Assay, Amplification, Electrophoretic Mobility Shift Assay

Journal: The Journal of Biological Chemistry

Article Title: Cancer-associated polybromo-1 bromodomain 4 missense variants variably impact bromodomain ligand binding and cell growth suppression

doi: 10.1016/j.jbc.2024.107146

Figure Lengend Snippet: Structural insights into the functional effects of cancer-associated PBRM1-BD4 missense variants. A , conserved residue Y580 stabilizes the loop-helix fold between the AB loop and the adjacent α B helix. B , frequently mutated residue R576 helps maintain the structural integrity of the α Z helix. C , frequently mutated residues M523 and M586 in the α Z and α B helices contribute to the stability of the PBRM1-BD4 α-helical core. D , conserved residue R540 contributes to the histone Kac binding pocket and adjacent α C helix stability. BD4, fourth bromodomain; PBRM1, polybromo-1.

Article Snippet: Sections of the coding region for PBRM1-containing BD4 mutations were synthesized (Biomatik) and inserted into the digested TetO-Fuw-PBRM1 WT plasmid (Addgene plasmid #85746) ( ) using the In-FusionHD cloning kit (Clontech Laboratories, Inc) and confirmed by WideSeq (Purdue University).

Techniques: Functional Assay, Residue, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Cancer-associated polybromo-1 bromodomain 4 missense variants variably impact bromodomain ligand binding and cell growth suppression

doi: 10.1016/j.jbc.2024.107146

Figure Lengend Snippet: Cancer-associated PBRM1-BD4 missense variants exhibit impaired stability and acetylated histone binding. A , immunoblot demonstrates equivalent expression of full-length PBRM1-BD4 missense variants in a Caki-2 tetracycline-inducible system. B , coimmunoprecipitation with BRG1 and V5-tagged PBRM1. C , immunoblots of V5-tagged PBRM1-BD4 WT and PBRM1-BD4 missense variants and beta-actin from Caki-2 cells treated with 100 μg/ml cycloheximide for 0, 2, 6, 10, or 24 h. D , immunoblot densitometry quantitation of PBRM1-BD4 WT and PBRM1-BD4 missense variants at 2 h of cycloheximide treatment. Significance was calculated using an unpaired Student’s t test, where ∗ p < 0.05, and error bars represent the SEM. E , correlation of PBRM1-BD4 WT and PBRM1-BD4 missense variant protein stability as assessed by the cellular cycloheximide chase assay at 2 h and the biophysical SYPRO Orange thermal shift assay, where horizontal error bars represent SD of immunoblot densitometry quantitation at 2 h of cycloheximide treatment and vertical error bars represent SD of protein T m values determined by the SYPRO Orange thermal shift assay. F , acetylated histone H3 peptide pulldown (n = 2) by PBRM1 WT and PBRM1-BD4 missense variants as measured by fold enrichment of H3K14,18,23,27ac(1–30) over input. Significance was calculated using an unpaired Student’s t test where ∗ p < 0.05 and error bars represent SEM. BD4, fourth bromodomain; BRG1, brahma-related gene 1; PBRM1, polybromo-1; T m , protein melting temperature.

Article Snippet: Sections of the coding region for PBRM1-containing BD4 mutations were synthesized (Biomatik) and inserted into the digested TetO-Fuw-PBRM1 WT plasmid (Addgene plasmid #85746) ( ) using the In-FusionHD cloning kit (Clontech Laboratories, Inc) and confirmed by WideSeq (Purdue University).

Techniques: Binding Assay, Western Blot, Expressing, Quantitation Assay, Variant Assay, Thermal Shift Assay

Journal: The Journal of Biological Chemistry

Article Title: Cancer-associated polybromo-1 bromodomain 4 missense variants variably impact bromodomain ligand binding and cell growth suppression

doi: 10.1016/j.jbc.2024.107146

Figure Lengend Snippet: Cancer-associated PBRM1-BD4 missense variants exhibit impaired cancer cell growth suppression and PBRM1 target gene regulation. A , workflow of the FACS cell proliferation competition assay. B , Caki-2 PBRM1 WT and PBRM1-BD4 missense variant cell growth over 22 days. Significant differences between cell lines were calculated using a two-way ANOVA (mixed model) with Tukey post hoc analysis where ∗ p < 0.05 and ∗∗ p < 0.01, and colored wedges around trendlines represent SD (n = 2 for vector, n = 4 for WT, n = 3 for missense variants). C , RT-qPCR (n = 3) of select PBRM1 target and nontarget genes. Significance was calculated using an unpaired Student’s t test where ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 and error bars represent the SEM. BD4, fourth bromodomain; FACS, fluorescence-activated cell sorting; PBRM1, polybromo-1; RT-qPCR, quantitative real-time PCR.

Article Snippet: Sections of the coding region for PBRM1-containing BD4 mutations were synthesized (Biomatik) and inserted into the digested TetO-Fuw-PBRM1 WT plasmid (Addgene plasmid #85746) ( ) using the In-FusionHD cloning kit (Clontech Laboratories, Inc) and confirmed by WideSeq (Purdue University).

Techniques: Competitive Binding Assay, Variant Assay, Plasmid Preparation, Quantitative RT-PCR, Fluorescence, FACS, Real-time Polymerase Chain Reaction