human brd4  (Addgene inc)

Journal: bioRxiv

Article Title: BRD4 represses developmental and neuronal genes through interactions with polycomb complexes

doi: 10.64898/2026.01.31.702994

Figure Lengend Snippet: a ) Dot plots showing log2 fold enrichment of BRD proteins in the proximal interactome (Turbo-ID) for PRC1 and PRC2 proteins from mouse embryonic stem cells (mESCs), data from . The size of the circle represents the log2 fold enrichment in BRD4 IP relative to IgG control. b ) Like (a) but for enrichment of PRC proteins in BRD4 immunoprecipitation from K562 cells, data from , . The size of the circle represents the t-test difference between the BRD4 IP and the IgG control. c) Immunoblots of endogenous BRD4 IP in H9 hESCs using antibodies that recognise both short and long BRD4 isoforms, with antibodies detecting RING1B, CBX7, CBX4, H3K27ac, H3K23ac, H3K27me3, along with reverse IP with RING1B and MGA antibodies followed by immunoblots for BRD4 and H3K27me3. d ) Immunoblots of GFP-trap co-immunoprecipitation of GFP-BRD4 long isoform (GFP-BRD4L) with Flag-tagged E2F6 and L3MBTL2, HA-tagged EED and EZH2. Immunoblots for β-ACTIN served as controls, e ) Heatmap of CUT&Tag for BRD4, EED, H3K23ac and ChIP-seq data for H3K14ac and RING1B, at active (H3K4me3+), bivalent (H3K4me3+/H3K27me3+) and PRC2 repressed promoters (H3K27me3+). f ) AlphaScreen counts titration of BRD4-BD1 and -BD2 interaction with H3K14ac/23ac showing that only BRD4-BD2 interacts with H3K14ac/23ac. Normalized average alpha counts of three replicates were set relative to the highest WT. g) Immunoblots of biotinylated H3K14/K23ac pulldown for N-terminal His-FLAG tagged BRD4 (N-terminal 412 amino acids), in the presence of increasing concentration of iBET-BD2 (iBD2).

Article Snippet: 1 μg of biotinylated histone H3K14ac/H3K23ac peptide (Cayman Chemicals, Cat. 27520-250ug-CAY) was incubated with 10 μL of streptavidin magnetic beads (Invitrogen 656-01) in 300 μL of binding buffer (50 mM Tris, pH 7.5, 200 mM NaCl and 0.1% NP-40, proteinase inhibitor cocktail) and rotated at room temperature for 30 min. At the same time, FLAG-His tagged BRD4 N -terminal domain containing BD1 and BD2 (E49-E460) (MedChemExpress Cat# HY-P7846), inhibitor of iBET-BD2 (Cayman Chemical Cat# CAY31766), or DMSO were added to the binding buffer on ice.

Techniques: Control, Immunoprecipitation, Western Blot, ChIP-sequencing, Amplified Luminescent Proximity Homogenous Assay, Titration, Concentration Assay

Journal: bioRxiv

Article Title: BRD4 represses developmental and neuronal genes through interactions with polycomb complexes

doi: 10.64898/2026.01.31.702994

Figure Lengend Snippet: a ) Heatmap showing BRD4 signal (CPM) for WT and BRD4 BD2 mut1 at protein-coding genes and active enhancers of hESCs. b ) Scatter plot comparing log2 fold change (log2 FC) values for BRD4 BD2-Mut1/WT (X-axis) against BRD4 dTAG/DMSO (Y-axis) conditions. GSEA GO-biological process enrichment lists for genes that are commonly up (red) and down (blue) regulated in both conditions (right). c ) Representative genome browser snapshot displaying signals for RNA-seq WT, BRD4-mutant1, DMSO and dTAGV-1 along with MAX, BRD4, H3K27me3 and H3K4me3. For CUT&Tag (BRD2,3,4, H3K4me3, H3K27me3) and CUT&Run (EED, ser5 Pol-II), the signal is compared as CPM and MAX as ChIP-seq signal from ChIP-atlas. d) Heatmaps displaying H3K27me3 and H3K4me3 ChIP-seq signals along with RNA-seq normalized counts at bivalent genes in WT-H9 and H9-derived BRD4 BD2 mut1 neurons. e ) MA plot illustrating differential gene expression in BRD4 BD2 mut1 compared to WT neurons. Significantly up- and down-regulated bivalent and non-bivalent genes are highlighted in red and blue, respectively. The number of differentially expressed genes with a log2 fold change of 1 and an adjusted p-value of <0.05 is indicated (right). f ) Genome browser tracks showing ChIP-seq data for bivalent histone modifications (H3K4me3 and H3K27me3), fold change over input and RNA-seq (RPKM) for neuronal genes.

Article Snippet: 1 μg of biotinylated histone H3K14ac/H3K23ac peptide (Cayman Chemicals, Cat. 27520-250ug-CAY) was incubated with 10 μL of streptavidin magnetic beads (Invitrogen 656-01) in 300 μL of binding buffer (50 mM Tris, pH 7.5, 200 mM NaCl and 0.1% NP-40, proteinase inhibitor cocktail) and rotated at room temperature for 30 min. At the same time, FLAG-His tagged BRD4 N -terminal domain containing BD1 and BD2 (E49-E460) (MedChemExpress Cat# HY-P7846), inhibitor of iBET-BD2 (Cayman Chemical Cat# CAY31766), or DMSO were added to the binding buffer on ice.

Techniques: RNA Sequencing, ChIP-sequencing, Derivative Assay, Gene Expression

Journal: bioRxiv

Article Title: BRD4 represses developmental and neuronal genes through interactions with polycomb complexes

doi: 10.64898/2026.01.31.702994

Figure Lengend Snippet: a) Schematic representation of the protocol used to generate unguided neuronal organoids (UNOs), with images of UNO WT at 5,8, and 41 days. b ) Immunofluorescence images of UNOs at day 41 stained for markers of neuronal progenitor (SOX2), post-mitotic early neurons (TUJ1), scale bars: 100 μm. c ) MA plot for RNA-seq data illustrating differentially expressed genes in day 41 UNOs following 20 hours of BRD4 PROTAC (ZxH) treatment (n=3 independent organoids). d) Geneontology (GO) enrichment analyses of up- and down-regulated genes. e ) Genome browser tracks for normalized reads at TSS for pseudo bulk scCUT&Tag and bulk RNA-seq for immediate early genes (IEGs) upon 20 h BRD4 PROTAC in UNOs (data from (c)). f) UMAP plots stratified by genotype show the annotated cell lineages: WT, BRD4 BD2 mut2, and BRD4 BD2 mut3. Cell clusters are identified by colour, illustrating the contribution of each genotype to specific lineages, such as Glutamatergic, GABAnergic, optic vesicle, and RPE. g) Stacked bar charts for 41-day and 63-day UNOs, detailing the percentage of cells for each annotated cell type across the WT, BRD4 BD2 mut2, and BRD4 BD2 mut3 UNOs. h) Representative bright-field microscopy images of 41-day UNOs, Scale bar=1mm (rest of the images in source file). i) Dot plots showing the average expression level (Z scores) and percentage of cells expressed in Glutamatergic, Diencephalic-1(pink in UMAP), and Diencephalic-2(blue in UMAP), and G2M clusters for bivalent genes that showed significant differential expression in the scRNA-seq data in BRD4-BD2 mut1 and BRD4-BD2 mut2 UNOs.

Article Snippet: 1 μg of biotinylated histone H3K14ac/H3K23ac peptide (Cayman Chemicals, Cat. 27520-250ug-CAY) was incubated with 10 μL of streptavidin magnetic beads (Invitrogen 656-01) in 300 μL of binding buffer (50 mM Tris, pH 7.5, 200 mM NaCl and 0.1% NP-40, proteinase inhibitor cocktail) and rotated at room temperature for 30 min. At the same time, FLAG-His tagged BRD4 N -terminal domain containing BD1 and BD2 (E49-E460) (MedChemExpress Cat# HY-P7846), inhibitor of iBET-BD2 (Cayman Chemical Cat# CAY31766), or DMSO were added to the binding buffer on ice.

Techniques: Immunofluorescence, Staining, RNA Sequencing, Microscopy, Expressing, Quantitative Proteomics

Journal: bioRxiv

Article Title: BRD4 represses developmental and neuronal genes through interactions with polycomb complexes

doi: 10.64898/2026.01.31.702994

Figure Lengend Snippet: a) UMAP plots show the distribution of single-cell ATAC sequencing (scATAC-seq) data clustered by genotypes WT and BRD4 BD2 mut2 and annotated by cell lineage for WT and BRD4 BD2 mut2. b ) Z-scores (high scores in red and low scores are in blue) showing top transcription factor motifs enriched at Diencephalic, Glutamatergic, G2M and GABAnergic lineages across scATACseq peaks, which are gained in BRD4 BD2 mut 2 UNO compared to WT control. The complete list of enriched TFs is in the source data table.

Article Snippet: 1 μg of biotinylated histone H3K14ac/H3K23ac peptide (Cayman Chemicals, Cat. 27520-250ug-CAY) was incubated with 10 μL of streptavidin magnetic beads (Invitrogen 656-01) in 300 μL of binding buffer (50 mM Tris, pH 7.5, 200 mM NaCl and 0.1% NP-40, proteinase inhibitor cocktail) and rotated at room temperature for 30 min. At the same time, FLAG-His tagged BRD4 N -terminal domain containing BD1 and BD2 (E49-E460) (MedChemExpress Cat# HY-P7846), inhibitor of iBET-BD2 (Cayman Chemical Cat# CAY31766), or DMSO were added to the binding buffer on ice.

Techniques: Sequencing, Control

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: (a) Cryo-EM map and (b) cartoon representation of the BRD4-S/nucleosome complex showing how the BRD4 BD1 (pink) interacts with the nucleosome. The modeled histone H4 tail residues 11-16 are shown as a thicker gold line.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: (a) representative motion-corrected micrographs, (b) representative 2D classes, (c) angular distribution of particles used to generate the cryo-EM map, (d) cryo-EM map of the BRD4/nucleosome complex colored by estimated local resolution determined with FSC = 0.143 cutoff in cryoSPARC, (e) conical Fourier shell correlation (cFSC) curve of the BRD4-nucleosome structure at 2.89 Å resolution, calculated between two independent half-maps using a conical mask with a specified half-angle and axis in Fourier space in cryoSPARC. Lines and arrows indicate the axis of rotation between successive views, (f) unmasked (red) and masked (blue) Fourier shell correlation (FSC) curves between two independent half-maps determined in cryoSPARC.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: Schematic representation of the cryo-EM data processing pipeline for the BRD4/nucleosome complex, as described in the Methods section.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: (a) cryo-EM map of viewed from the side to show interaction of histone H4 tail with BRD4 BD1, (b) cryo-EM map prepared from subset of particles shows extra density (purple) beyond the C-terminus of BRD4 BD1 (top view on left, side view on right).

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: (a) Time-resolved FRET binding assay results for BRD4-S binding to unmodified (blue), H4 tailless = H4(24-102) (green) and H4 K12acK16ac nucleosomes (pink) in 70 mM NaCl. (b) Effect of NaCl concentration on BRD4-S binding to unmodified (blue) or H4 K12acK16ac nucleosomes (pink) as assayed by TR-FRET.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques: Binding Assay, Concentration Assay

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: (a) top: cartoon representation of BRD4 constructs, bottom: individual TR-FRET unnormalized fluorescence (not normalized to maximum fluorescence) binding results of for wild-type BRD4-S binding to H4 K12acK16ac nucleosomes and average of 3 measurements for BRD4 BD1 and BD2 binding to H4 K12acK16ac nucleosomes, (b) TR-FRET binding curves for BRD4-S binding to unmodified (blue) or H4 K12acK16ac (pink) nucleosomes in 100, 125 and 150 mM NaCl.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques: Construct, Fluorescence, Binding Assay

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: (a) BRD4-S domains and basic patches highlighted in cartoon and primary sequence (left) and identity of BRD4-S basic patch mutations studied (right), (b) TR-FRET dissociation constants for BRD4-S basic patch mutants binding to H4 K12acK16ac nucleosomes in 70 mM NaCl, (c) TR-FRET dissociation constants for BRD4-S basic patch mutants binding to H4 K12acK16ac nucleosomes in 150 mM NaCl, (d) effect of salt concentration on select BRD4-S basic patch mutations on binding to H4 K12acK16ac nucleosomes.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques: Sequencing, Binding Assay, Concentration Assay

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet:

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques:

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: (a) BRD4 BD1 interactions with the nucleosome with key regions highlighted, (b) TR-FRET dissociation constants for BRD4-S bromodomain mutants binding to H4 K12acK16ac nucleosomes, (c) model for how BRD4 basic region 1 could interact with nucleosome DNA minor groove with C ⍺ positions of the 5 basic residues shown in blue spheres, (d) effect of BRD4-S BD1 mutations on BRD4-S/nucleosome complex mobility in gel mobility shift assay, (e) NMR structure of HMG-I(Y) AT-hook Arg-Gly-Arg region binding to DNA (PDB ID 2EZD), protein residues outside of the Arg-Gly-Arg region not shown, (f) TR-FRET dissociation constants for BRD4-S basic patch 1 mutants binding to H4 K12acK16ac nucleosomes.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques: Binding Assay, Mobility Shift

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: Acidic patch nucleosomes contain the H2A(E61A,E64A,D90A,E92A) quadruple mutation. BRD4-S binds wild-type nucleosomes (blue) and acidic patch nucleosomes (red) with similar affinity, but the RCC1 chromatin factor shown to use an arginine anchor to bind to the nucleosome acidic patch is adversely affected by the nucleosome acidic patch mutations.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: (a) the BRD4-S m12/H4 K12acK16ac nucleosome complex (blue) elutes earlier than the BRD4-S WT/H4 K12acK16ac nucleosome complex, (b) the BRD4-S m12 protein elutes at similar time as the BRD4 WT protein.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques:

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: The BRD4 residues R68, K72, K76 observed to interact with nucleosomal DNA and the BRD2, BRD3 and BRDT equivalent BD1 and BD2 residues are highlighted in yellow.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques:

Journal: bioRxiv

Article Title: BRD4 binds the nucleosome via both histone and DNA interactions

doi: 10.1101/2025.05.29.656846

Figure Lengend Snippet: Superposition of ZL0590/BRD4 BD1 crystal structure (PDB 6U0D) with BRD4/nucleosome structure (this work) via BRD4 BD1.

Article Snippet: The coding region of the short isoform of human BRD4 (residues 1–722) was amplified from the plasmid pGEX-6P-1 BRD4 full-length (a gift from Peter Howley, AddGene plasmid #14447 ) and cloned into the pST50Tr-HISN expression vector .

Techniques:

Journal: The FEBS journal

Article Title: Gα s -specific structural elements attenuate interactions with regulator of G protein signaling (RGS) proteins.

doi: 10.1111/febs.70149

Figure Lengend Snippet: Fig. 1. Gas GTPase activity is not affected by RGS16. (A) Representative single-turnover GTPase assays of Gas (400 nM), without RGS16 (open blue circles) and with 2 lM RGS16 (blue circles). The average (n = 3) GTPase rate constant (K ) was 1 0.05 min1 for Gas in the presence of 2 lM RGS16 and 1.2 0.2 min1 for the basal activity of Gas. (B) Representative single-turnover GTPase assays of Gai1 (400 nM), in the presence of 20 nM RGS16 (green triangles) and without added RGS16 (open green triangles). The average (n = 3) reaction rate constant (k) was 2.2 0.2 min1 in the presence of RGS16 (20 nM), while the basal activity of Gai1 was 0.3 0.05 min1. (C) Representative dose–response analysis of RGS16 activity toward wild-type Gas (blue circles) and wild-type Gai1 (green triangles, EC50 = 5 1 nM). Data presented are mean sem of experiments performed in triplicate, representative of three independent biological replicates each. Reaction rate constants for single-turnover GTPase assays were calculated using a single-exponential fit to the data using SigmaPlot 10.0, while EC50 values for dose–response analysis of the net RGS-induced GTPase activity were calculated using three parameter sigmoidal curves.

Article Snippet: The RGS domain of human RGS16 (residue 86–205) and human Gai1 (residues 31–354) were expressed in the pNIC-SGC1 vector (Addgene, Watertown, MA, USA) and pProEXHTb vector (ThermoFisher Scientific, Waltham, MA, USA), respectively.

Techniques: Activity Assay

Journal: The FEBS journal

Article Title: Gα s -specific structural elements attenuate interactions with regulator of G protein signaling (RGS) proteins.

doi: 10.1111/febs.70149

Figure Lengend Snippet: Fig. 2. Structural elements in the Gas helical domain that may disrupt interactions with RGS domains. (A) Alphafold2 models of wild-type Gas, modeled bound to RGS16. The Gas-RGS16 complex modeled using the crystal structure of the Gai1-RGS16 complex (PDB ID: 2IK8) by superimposing the two Ga subunits. The five models of Gas produced by Alphafold2 using the ColabFold server are shown in tube representation, colored teal (GTPase domain) and blue (helical domain). Five Gas-unique insertions, four in the GTPase domain and one in the helical domain, that are far away from the RGS domain, are colored green. Two Gas regions that contain Gas-unique insertions that are close to the putative location of the RGS domain and may introduce a steric clash are the aB-aC loop (V134-D141, red) and the a1-aA region (G67-D85, shades of magenta). The a1-aA region showed a variable conformation among the Alphafold2 models. (B) Representative experimental structures of Ga subunits that interact with high-activity RGS proteins (Gai1, Gao, Gaq). Ga subunits from five representative crystal structures (PDB IDs 2GTP, 1AGR, 2IK8, 3C7K, 4EKD) were superimposed, with Ga subunits shown as tubes colored orange (GTPase domain) and gold (helical domain). Ga aB-aC loops are colored in shades of blue and the a1-aA regions are colored cyan. In both panels, RGS16 from the Gai1–RGS16 complex (2IK8) is shown as a transparent gray molecular surface. 3D structural visualizations were carried out with the molecular graphics program PyMol.

Article Snippet: The RGS domain of human RGS16 (residue 86–205) and human Gai1 (residues 31–354) were expressed in the pNIC-SGC1 vector (Addgene, Watertown, MA, USA) and pProEXHTb vector (ThermoFisher Scientific, Waltham, MA, USA), respectively.

Techniques: Produced, Introduce, Activity Assay

Journal: The FEBS journal

Article Title: Gα s -specific structural elements attenuate interactions with regulator of G protein signaling (RGS) proteins.

doi: 10.1111/febs.70149

Figure Lengend Snippet: Fig. 3. Specific Gas structural elements that can modulate interactions with RGS proteins. Multiple sequence alignment of Gas, Gai1, Gao, and Gaq, aligned using MAFFT and adjusted manually based on structure alignment of the representative structures of the Ga subunits (Fig. 2). Sequences were taken from the UNIPROT database as follows: Gas (P63092-1), Gai1 (P63096), Gao (P09471), and Gaq (P50148). The helical domain insertions identified in Fig. 2 as putative disruptor elements are highlighted in magenta, while putative permissive insertions are highlighted in green. Residues that contribute to Gai/o/q interaction with RGS proteins according to our previous energy calculations [37,45] are marked under the alignment with asterisks. Black asterisks mark residues that are not unique to Gas, while blue asterisks mark Gas-unique residues that correspond to contributing Gai/o/q positions. Putative Gas disruptor residues, which clash or introduce an unfavorable electrostatic interaction with RGS residues, are highlighted in red. The helical domain and the three switch (Sw) regions are marked above the alignment. The N- and C- termini of the Ga subunits (~40 and ~30 residues, respectively), which are distant from the RGS domains, were truncated for brevity.

Article Snippet: The RGS domain of human RGS16 (residue 86–205) and human Gai1 (residues 31–354) were expressed in the pNIC-SGC1 vector (Addgene, Watertown, MA, USA) and pProEXHTb vector (ThermoFisher Scientific, Waltham, MA, USA), respectively.

Techniques: Sequencing, Introduce

Journal: The FEBS journal

Article Title: Gα s -specific structural elements attenuate interactions with regulator of G protein signaling (RGS) proteins.

doi: 10.1111/febs.70149

Figure Lengend Snippet: Fig. 4. Structural elements in the Gas GTPase domain that may disrupt interactions with RGS domains. (A) Model of the Gas GTPase domain with RGS16. Gas from PDB ID 1AZS and Gai1 with RGS16 from PDB ID 2IK8 were superimposed with PyMol using the coordinates of the Ga subunits. The three Gas residues that may disrupt interaction with the RGS domain are shown as red spheres. Gas and Gai1 are shown in tube representation. Gas is colored teal (GTPase domain) and blue (helical domain), and Gai1 is colored orange (GTPase domain) and gold (helical domain). Ga structures are rotated 30° about the X-axis relative to Fig. 2, with the Ga helical domain and residues surrounding the highlighted residues omitted for clarity. (B) Close-up view of the predicted unfavorable interactions of the three putative Gas disruptor residues shown in A (red sticks) with the RGS16 residues with whom they interact unfavorably (gray sticks). Atoms that have a steric clash between them in the model are marked with transparent spheres. Predicted unfavorable electrostatic interactions between Gas D229 and RGS16 E129, which are ~1.5 A in this model, are marked with dashed red lines. (C) The corresponding three Gai1 residues, in relation to the same RGS16 residues shown in B. 3D structural visualizations were carried out with the molecular graphics program PyMol.

Article Snippet: The RGS domain of human RGS16 (residue 86–205) and human Gai1 (residues 31–354) were expressed in the pNIC-SGC1 vector (Addgene, Watertown, MA, USA) and pProEXHTb vector (ThermoFisher Scientific, Waltham, MA, USA), respectively.

Techniques:

Journal: The FEBS journal

Article Title: Gα s -specific structural elements attenuate interactions with regulator of G protein signaling (RGS) proteins.

doi: 10.1111/febs.70149

Figure Lengend Snippet: Fig. 5. Substituting Gai1 GTPase domain residues with the corresponding Gas putative disruptor residues is sufficient to abolish RGS16 GAP activity. Representative dose–response analysis of RGS16 activity toward wild-type Gai1 (green triangles), Gai1-V185F single mutant (open orange triangles and dashed line), Gai1-S206D single mutant (open red squares), Gai1-H213Q single mutant (orange circles), Gai1-V185F/H213Q double mutant (open orange circles), and Gai1-V185F/S206D/H213Q triple mutant (magenta squares). EC50 values are as follows: wild-type Gai1 = 5 1 nM (n = 3), Gai1-V185F single mutant = 5 1 nM (n = 3), Gai1-S206D single mutant (n = 3), Gai1-H213Q single mutant = 9 3 nM (n = 5), Gai1-V185F/H213Q double mutant = 40 10 nM (n = 5), and Gai1-V185F/S206D/H213Q triple mutant (n = 3). Data presented are mean sem of experiments performed in triplicate with the number of biological replicates reported in parentheses. EC50 values of net RGS-induced GTPase activities were calculated using three parameter sigmoidal curves with SigmaPlot 10.

Article Snippet: The RGS domain of human RGS16 (residue 86–205) and human Gai1 (residues 31–354) were expressed in the pNIC-SGC1 vector (Addgene, Watertown, MA, USA) and pProEXHTb vector (ThermoFisher Scientific, Waltham, MA, USA), respectively.

Techniques: Activity Assay, Mutagenesis

Journal: The FEBS journal

Article Title: Gα s -specific structural elements attenuate interactions with regulator of G protein signaling (RGS) proteins.

doi: 10.1111/febs.70149

Figure Lengend Snippet: Fig. 6. Substituting all three putative Gas disruptor residues with the corresponding Gai1 residues results in full RGS16 gain of function. Representative dose–response analysis of RGS16 activity toward wild-type Gas (blue circles), Gas-D229S single mutant (open red squares), Gas-F208V/Q236H double mutant (open orange circles), and Gas-F208V/D229S/Q236H triple mutant (magenta squares). EC50 values are as follows: wild-type Gas = N/A (n = 3), Gas D229S single mutant = 900 150 nM (n = 4), Gas-F208V/ Q236H double mutant = N/A (n = 3), and Gas F208V/D229S/Q236H triple mutant = 10 2 nM (n = 4). Data presented are mean sem of experiments performed in triplicate with the number of biological replicates reported in parentheses. EC50 values of net RGS-induced GTPase activities were calculated using three parameter sigmoidal curves with SigmaPlot 10.

Article Snippet: The RGS domain of human RGS16 (residue 86–205) and human Gai1 (residues 31–354) were expressed in the pNIC-SGC1 vector (Addgene, Watertown, MA, USA) and pProEXHTb vector (ThermoFisher Scientific, Waltham, MA, USA), respectively.

Techniques: Activity Assay, Mutagenesis

Journal: Nature Communications

Article Title: Zfp260 choreographs the early stage osteo-lineage commitment of skeletal stem cells

doi: 10.1038/s41467-024-54640-0

Figure Lengend Snippet: a Workflow of in vivo labeling strategy using TAM and the experimental design. b PCA indicating the variations of transcriptomes among Lin - ZsGreen + cells isolated from BF-Ctrl, BF-cKO, MSFL-Ctrl and MSFL-cKO groups. c GO and KEGG enrichment analysis of FACS-RNA-seq data. d Heatmap of replicate data for H3K4me1 and H3K27ac enrichment as detected by CUT&Tag. n = 3 from 3 biological replicates. e GO-biological process enrichment analysis of differentially enriched super-enhancers. f Heatmap of replicate data for Zfp260-V5 enrichment detected by ChIP-seq. n = 2 from 2 biological replicates. g Top enriched de novo motifs of Zfp260-V5 enriched genes. h Distribution of peaks in the genome. i GO and KEGG enrichment analysis of Zfp260-V5 enriched genes. j Screening strategy for the potential master downstream regulator. k Transcripts Per Kilobase (TPM) of Runx2 expression level from fracture and MSFL derived Lin - ZsGreen + cells. n = 3 from 3 biological replicates of RNA-seq data. l Genome browser view of peaks enriched for H3K4me1, Brd4, H3K27ac, and Zfp260-V5 over the Runx2 gene locus on chromosome 17 (left) with the magnified super-enhancer region displayed on the right. Primers 1 and 2 indicated the primer sets for the subsequent ChIP-qPCR detection. m Co-IP was performed to examine the condensates for the super-enhancer via immortalized PSCs. n = 3 from 3 biological replicates. n , o mIHC co-staining for Zfp260 (purple) with Brd4 (gold), Med1 (cyan), and P300 (gray) in the homeostatic and osteogenic states of PSCs. The yellow dotted line indicated the route for the subsequent fluorescence intensity measurements. n = 3 from 3 biological replicates. p Fluorescence intensity measurements along the route, with black triangles indicating the merged signals of the four channels. q , r ChIP-qPCR assays for H3K27ac and Brd4 binding via immortalized PSCs. n = 6 from 2 biological replicates. Two-way ANOVA. Scalebars: 5 μm. All data in this figure are represented as mean ± SD. Source data and exact p values are provided in the Source Data file.

Article Snippet: The primary antibodies used in mIHC (dilution 1:400 for all antibodies) included goat anti-mouse/human/rat Itgav (AF1219, Novus Biologicals), mouse anti-mouse/rat CD90 (NB100-65543, Novus Biologicals), mouse anti-mouse/human CD105 (NBP2-22122, Novus Biologicals), rabbit anti-human/mouse/rat CD200 (AF2724, Novus Biologicals), rabbit anti-mouse/human/rat Runx2 (ab236639, Abcam), rabbit anti-mouse/human/rat Sox9 (ab185966, Abcam), rabbit anti-mouse/human Alpl (MA5-24845, Invitrogen), rabbit anti-mouse/human/rat Zfp260 (ABE295, Merck), mouse anti-human/mouse/rat p300 (NB100-616, Novus Biologicals), rabbit anti-human/mouse MED1 (NB100-2574, Novus Biologicals), rabbit anti-human/mouse BRD4 (NBP2-76393, Novus Biologicals), mouse anti-human/mouse/rat Prkca (NB600-201, Novus Biologicals), rabbit anti-V5 tag (13202, CST), mouse anti-Collagen type I (67288-1-Ig, proteintech), rabbit anti-Collagen type II (28459-1-AP, proteintech).

Techniques: In Vivo, Labeling, Isolation, RNA Sequencing Assay, ChIP-sequencing, Expressing, Derivative Assay, Co-Immunoprecipitation Assay, Staining, Fluorescence, Binding Assay

Journal: Nature Communications

Article Title: Zfp260 choreographs the early stage osteo-lineage commitment of skeletal stem cells

doi: 10.1038/s41467-024-54640-0

Figure Lengend Snippet: a GST-pull down assay of PSC’s whole cell lysate (WCL). The red dotted box indicated the regions for M/S analysis. b GST-Zfp260 specially enriched kinases with high HT sequest scores. c Co-IP of 293 T cell line. d Co-IP of immortalized PSCs. e Co-staining image for Zfp260 and Prkca in PSCs. The white dotted circle indicated the nucleus (Left). The white dotted line indicated the route for the fluorescence intensity measurements (right). f Representative images of immunofluorescence of Zfp260 with osteogenic induction, with the MFI/cytosolic MFI calculated (right). g Separation of nuclear (NE) and cytosolic extracts (CE) followed by Western blot. h In vitro Phos-Assay was performed by Phospho-PAGE. i Co-IP, Phos-PAGE, and SDS-PAGE were jointly performed in immortalized PSCs. The red dotted rectangle indicating the Y173, S182, and S197 residues. j Suggested binding mode of Zfp260 and Prkca by AlphaFold2. k The shortest distance between Zfp260-aa173 and the catalytic domain (CD) of Prkca (Z173-CD), aa182 and CD (Z182-CD), and aa197 and CD (Z197-CD) were calculated. l Co-IP was performed in immortalized PSCs, with statistical analysis (right). m Representative images of immunofluorescence of Zfp260-V5 before and after osteogenic induction (left), with the nuclear MFI/cytosolic MFI evaluated (right). n Separation of NE and CE, followed by Western blot. o Representative ARS staining images of PSCs. Scalebar: 2 mm. p–r ChIP-qPCR assays for Zfp260-V5, Brd4, and H3K27ac binding. n = 6 from 2 biological replicates. For ( a , d , e , h , i ), experiments were conducted independently 3 times, consistently producing similar results. For ( c , g , h , i , l , n , o ), n = 3 from 3 biological replicates. For ( f , m ), n = 30 from 3 biological replicates, with 10 randomly selected cells calculated per replicate. For ( e , f , m ), scale bars: 5 μm. Two-way ANOVA. Box plots display the minimum and maximum values, with the center line representing the median, and the bounds of the box representing the 25th to 75th percentiles. Other data in this figure are represented as mean ± SD. Source data and exact p values are provided in the Source Data file.

Article Snippet: The primary antibodies used in mIHC (dilution 1:400 for all antibodies) included goat anti-mouse/human/rat Itgav (AF1219, Novus Biologicals), mouse anti-mouse/rat CD90 (NB100-65543, Novus Biologicals), mouse anti-mouse/human CD105 (NBP2-22122, Novus Biologicals), rabbit anti-human/mouse/rat CD200 (AF2724, Novus Biologicals), rabbit anti-mouse/human/rat Runx2 (ab236639, Abcam), rabbit anti-mouse/human/rat Sox9 (ab185966, Abcam), rabbit anti-mouse/human Alpl (MA5-24845, Invitrogen), rabbit anti-mouse/human/rat Zfp260 (ABE295, Merck), mouse anti-human/mouse/rat p300 (NB100-616, Novus Biologicals), rabbit anti-human/mouse MED1 (NB100-2574, Novus Biologicals), rabbit anti-human/mouse BRD4 (NBP2-76393, Novus Biologicals), mouse anti-human/mouse/rat Prkca (NB600-201, Novus Biologicals), rabbit anti-V5 tag (13202, CST), mouse anti-Collagen type I (67288-1-Ig, proteintech), rabbit anti-Collagen type II (28459-1-AP, proteintech).

Techniques: Pull Down Assay, Co-Immunoprecipitation Assay, Staining, Fluorescence, Immunofluorescence, Western Blot, In Vitro, SDS Page, Binding Assay