gst tagged human brd4  (Addgene inc)

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 1. BRD4 NCC loss of function produces severe craniofacial phenotypes.

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques:

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 2. BRD4 mutant mandibular cNCCs fail to properly differentiate to osteoblast lineages.

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques: Mutagenesis

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 3. Loss of BRD4 disrupts in vitro cNCCs osteoblast differentiation.

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques: In Vitro

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 4. BRD4 binds to proximal active enhancers to regulate osteogenic transcription.

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques:

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 5. BRD4 directly regulates transcription of factors critical for osteoblast differentiation.

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques:

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 6. BRD4 associates with RUNX2 to regulate osteoblast differentiation

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques:

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 7. Model of BRD4 function in CdLS craniofacial pathogenesis (created with

Article Snippet: GST tagged human BRD4 (addgene 14447) was modified to delete the N-terminal portion by BamHI/BsaBI restriction digest and Gibson assembly to fuse BRD4 from amino acid E653-F1362 in frame (GST-BRD4-C-term) or out of frame (GST control) with GST.

Techniques:

Journal: bioRxiv

Article Title: Targeting fungal BET bromodomains as a pan - Candida antifungal strategy

doi: 10.1101/2023.02.03.527073

Figure Lengend Snippet: ( a ) Domain boundaries of Cg BDF1 BDs. ( b ) Phylogeny of fungal and human BET BDs. ( c ) Summary of binding intensities of WT and mutant forms (Y166F or Y343F) of Cg BDF1 BD1 and BD2 to an array of acetylated H3 and H4 peptides. Binding intensities are normalized to a positive control signal, obtained from the interaction of an anti-c-myc antibody with a c-myc control peptide included on the array. ( d ) Pull-down assay. Immobilized H4ac0 and H4ac4 peptides were incubated with GST-tagged Cg Bdf1 BDs or with the corresponding Y/F mutants. After washing with buffers containing either 150, 300 or 500 mM NaCl, bound proteins were eluted and visualized by western blotting with an anti-GST antibody. ( e ) Colony formation assays showing the effect of Bdf1 repression or Bdf1 BD inactivation on C. glabrata growth.

Article Snippet: GST-tagged human Brd4 BD2 (residues 349–460) was purchased from Reaction Biology Corp. Human Brd4 BD1 (residues 22–204), CaBdf1 BD1 (residues 193–327) and BD2 (residues 361–501), CgBdf1 BD1 (residues 120–248) and BD2 (residues 289–411) and CauBdf1 BD1 (residues 127–257) and BD2 (residues 287–416) were cloned into a pGEX4t1 vector as GST-tagged fusion proteins.

Techniques: Binding Assay, Mutagenesis, Positive Control, Pull Down Assay, Incubation, Western Blot

Journal: bioRxiv

Article Title: Targeting fungal BET bromodomains as a pan - Candida antifungal strategy

doi: 10.1101/2023.02.03.527073

Figure Lengend Snippet: ( a ) Overview of the HTRF assay. A GST-tagged BET BD is bound to an anti-GST antibody coupled to the donor fluorophor (Lumi4-Terbium cryptate). A biotinylated H4ac4 peptide is bound to streptavidin coupled to the acceptor fluorophore (D2 dye). The binding of GST-BD to H4ac4 brings the donor and acceptor into close proximity, allowing for FRET. Following donor excitation, fluorescence emitted by the acceptor is detected following a delay between the excitation pulse and the time-gated measurement window. The presence of a BET inhibitor disrupts the binding interaction, resulting in reduced HTRF signal. ( b ) HTRF assays performed on Cg Bdf1 BD1 and BD2 (closed purple and open magenta circles, respectively) and human Brd4 BD1 and BD2 (closed dark green and open light green circles, respectively) in the presence of the indicated compound. IC 50 values are listed next to each graph. Data represent the mean and s.d. values from three independent experiments. ( c ) Potency and selectivity of BETi compounds. Top. Summary of p IC 50 values showing the potency of compounds against Brd4 and Cg Bdf1 BDs. Symbol definitions are as in (b). Bottom. Summary of Δp IC 50 values showing the selectivity of inhibitors towards the human BDs. Closed blue (BD1) and open cyan (BD2) circles represent the difference in p IC 50 value between corresponding Brd4 and Cg Bdf1 BDs. ( d ) The four BET inhibitors do not affect C. glabrata growth, even when Bdf1 BD1 or BD2 is deleted. Expression of endogenous BDF1 was suppressed by a 23h incubation with methionine and cysteine and hence only the WT or mutant Bdf1 protein encoded by the plasmid was expressed. Strains were then incubated with BETi compounds at 10 μM for 24h and viability was assessed. Data represent the mean and s.d. values from three independent experiments.

Article Snippet: GST-tagged human Brd4 BD2 (residues 349–460) was purchased from Reaction Biology Corp. Human Brd4 BD1 (residues 22–204), CaBdf1 BD1 (residues 193–327) and BD2 (residues 361–501), CgBdf1 BD1 (residues 120–248) and BD2 (residues 289–411) and CauBdf1 BD1 (residues 127–257) and BD2 (residues 287–416) were cloned into a pGEX4t1 vector as GST-tagged fusion proteins.

Techniques: HTRF Assay, Binding Assay, Fluorescence, Expressing, Incubation, Mutagenesis, Plasmid Preparation

Journal: Molecular cell

Article Title: BET Bromodomain Proteins Function as Master Transcription Elongation Factors Independent of CDK9 Recruitment

doi: 10.1016/j.molcel.2017.06.004

Figure Lengend Snippet: (A) Heatmap of BRD4-nluc fusion protein levels normalized to fluc control levels in either 293FTWT or 293FTCRBN−/− cells. Results of 10-point dose-response experiment (n = 4) after 4 hr of drug incubation are summarized as AUC (area under the curve).

Article Snippet: BRD4 (49-170) GST tag human , Sigma Aldrich , Cat#SRP0459.

Techniques: Incubation

Journal: Molecular cell

Article Title: BET Bromodomain Proteins Function as Master Transcription Elongation Factors Independent of CDK9 Recruitment

doi: 10.1016/j.molcel.2017.06.004

Figure Lengend Snippet: (A) Rank ordered heatmap of H3K27ac (gray) and BRD4 levels at transcriptional start sites (TSS) after treatment with 1 µM JQ1 (blue), 100 nM dBET6 (red), or DMSOas vehicle control (black). Each row shows ± 5 kb centered on BRD4 peak. Rows are ordered by max BRD4 in each region (ranking based on DMSO). ChIP-Rx signal (rrpm) is depicted by color-scaled intensities. The ChIP-Rx signal was normalized by spike-in controls.

Article Snippet: BRD4 (49-170) GST tag human , Sigma Aldrich , Cat#SRP0459.

Techniques:

Journal: Molecular cell

Article Title: BET Bromodomain Proteins Function as Master Transcription Elongation Factors Independent of CDK9 Recruitment

doi: 10.1016/j.molcel.2017.06.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: BRD4 (49-170) GST tag human , Sigma Aldrich , Cat#SRP0459.

Techniques: Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Cell Viability Assay, Amplified Luminescent Proximity Homogenous Assay, Isolation, Kinase Assay, Expressing, Generated, Transduction, Plasmid Preparation, Sequencing, Software