chip grade antibodies for brd4  (Bethyl)

Journal: Cell Death & Disease

Article Title: Bromodomain-containing protein 4 (BRD4) as an epigenetic regulator of fatty acid metabolism genes and ferroptosis

doi: 10.1038/s41419-022-05344-0

Figure Lengend Snippet: Sequence for primers.

Article Snippet: Then, the fragments were precipitated following the kit manufacturer’s instructions using the BRD4 ChIP grade antibody (Abcam, Cat. No. Ab243862) (CST, Cat. No.9003).

Techniques: Sequencing

Journal: Cell Death & Disease

Article Title: Bromodomain-containing protein 4 (BRD4) as an epigenetic regulator of fatty acid metabolism genes and ferroptosis

doi: 10.1038/s41419-022-05344-0

Figure Lengend Snippet: Sequence for shRNA, siRNA and sgRNA.

Article Snippet: Then, the fragments were precipitated following the kit manufacturer’s instructions using the BRD4 ChIP grade antibody (Abcam, Cat. No. Ab243862) (CST, Cat. No.9003).

Techniques: Sequencing, shRNA

Journal: Cell Death & Disease

Article Title: Bromodomain-containing protein 4 (BRD4) as an epigenetic regulator of fatty acid metabolism genes and ferroptosis

doi: 10.1038/s41419-022-05344-0

Figure Lengend Snippet: Antibody working information.

Article Snippet: Then, the fragments were precipitated following the kit manufacturer’s instructions using the BRD4 ChIP grade antibody (Abcam, Cat. No. Ab243862) (CST, Cat. No.9003).

Techniques:

Journal: Cell Death & Disease

Article Title: Bromodomain-containing protein 4 (BRD4) as an epigenetic regulator of fatty acid metabolism genes and ferroptosis

doi: 10.1038/s41419-022-05344-0

Figure Lengend Snippet: A Relative BRD4 protein expression levels detected by western blotting ( n = 3, Mean ± SD, Tukey–Kramer test of one-way ANOVA). B Cell viability analysis through the MTT assay ( n = 5, Mean ± SD, Dunnett’s test of one-way ANOVA). C Relative protein expression levels tested by western blotting ( n = 3, Mean ± SD, Tukey–Kramer test of one-way ANOVA). D Intracellular lipid ROS of each group, determined by the boron-dipyrromethene C-11 probe. Green fluorescence reflects the total number of cells, red fluorescence indicates lipid ROS and the merged image represents the distribution intensities of lipid ROS in cells. E Intracellular Fe 2+ content tested by Prussian-blue staining. F and I Cell viability analysis through the MTT assay ( n = 5, Mean ± SD, Dunnett’s test of one-way ANOVA). Relative mRNA and protein expression levels of BRD4 detected by qRT-PCR ( G ) and western blotting ( H ) ( n = 3, Mean ± SD, Tukey–Kramer test of one-way ANOVA).

Article Snippet: Then, the fragments were precipitated following the kit manufacturer’s instructions using the BRD4 ChIP grade antibody (Abcam, Cat. No. Ab243862) (CST, Cat. No.9003).

Techniques: Expressing, Western Blot, MTT Assay, Fluorescence, Staining, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Bromodomain-containing protein 4 (BRD4) as an epigenetic regulator of fatty acid metabolism genes and ferroptosis

doi: 10.1038/s41419-022-05344-0

Figure Lengend Snippet: A Pie chart of the distribution of BRD4-bound chromatin fragments. B Analysis of BRD4 binding region distribution in chromatin. C Gene enrichment pathways corresponding to BRD4-bound chromatin fragments. D Gene enrichment analysis results of the GSH synthesis, iron metabolism, and lipid metabolism pathways from KEGG, Reactome , and WikiPathway database, respectively. E Genes involved in fatty acid metabolism pathways reflected in all ( KEGG, Reactome and WikiPathway ) databases. F Maps of HADH, ACSL1, and ACAA2 from KEGG pathway database. In C and D , the scatterplot is a graphical grouping display method based on the results of the gene enrichment analysis. GeneRatio refers to the ratio between the number of genes enriched in the term and the number of annotated genes. P. adjust refers to the p -value of each term calculated using the Fisher test (the probability of hypothesis test). The smaller the p -value, the greater the significance and the lower the misjudgment rate. In F , red boxes represent the metabolic steps involved in HADH, ACSL1 and ACAA2.

Article Snippet: Then, the fragments were precipitated following the kit manufacturer’s instructions using the BRD4 ChIP grade antibody (Abcam, Cat. No. Ab243862) (CST, Cat. No.9003).

Techniques: Binding Assay

Journal: Cell Death & Disease

Article Title: Bromodomain-containing protein 4 (BRD4) as an epigenetic regulator of fatty acid metabolism genes and ferroptosis

doi: 10.1038/s41419-022-05344-0

Figure Lengend Snippet: A The enrichment of BRD4, H3K27ac, P300, and Pol II in the promoter region of HADH, ACSL1, and ACAA2 via the ChIP/ qRT-PCR assay. Transcription activity of BRD4 on HADH, ACSL1, and ACAA2 promoters regions via dual-luciferase assay ( B ) and yeast one-hybrid system C . ( n = 3, Mean±SD, one-way ANOVA, * P < 0.05; ** P < 0.01; *** P < 0.005; N.S.: not significant). The mRNA ( D ) and protein ( E ) expression levels of HADH, ACSL1, and ACAA2 detected by qRT-PCR and Western blotting ( n = 3, Mean ± SD, one-way ANOVA, * P < 0.05; ** P < 0.01; *** P < 0.005; N.S.: no significant). F Co-localization analysis between HADH/ ACSL1/ ACAA2 and the mitochondria.

Article Snippet: Then, the fragments were precipitated following the kit manufacturer’s instructions using the BRD4 ChIP grade antibody (Abcam, Cat. No. Ab243862) (CST, Cat. No.9003).

Techniques: Quantitative RT-PCR, Activity Assay, Luciferase, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Bromodomain-containing protein 4 (BRD4) as an epigenetic regulator of fatty acid metabolism genes and ferroptosis

doi: 10.1038/s41419-022-05344-0

Figure Lengend Snippet: A Networks of SEs that bind to BRD4 and associated with HADH, ACSL1, and ACAA2. B Genomic locations of SEs. C Motif of BRD4 binding sites. D The enrichment of BRD4 in the genomic regions of intergenic lncRNAs that near the target mRNA genes via the ChIP/ qRT-PCR assay. E , F Relative mRNA levels detected by qRT-PCR ( n = 3, Mean ± SD, one-way ANOVA, * P < 0.05; ** P < 0.01; *** P < 0.005). G The predicted framework of fatty acid metabolism regulated by BRD4 in mitochondria.

Article Snippet: Then, the fragments were precipitated following the kit manufacturer’s instructions using the BRD4 ChIP grade antibody (Abcam, Cat. No. Ab243862) (CST, Cat. No.9003).

Techniques: Binding Assay, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Bromodomain-containing protein 4 (BRD4) as an epigenetic regulator of fatty acid metabolism genes and ferroptosis

doi: 10.1038/s41419-022-05344-0

Figure Lengend Snippet: A The co-IP silver staining diagram. B Heat map showing ignificantly down-regulated binding proteins. C Molecular function (MF) of significantly down-regulated binding proteins through GO enrichment analysis. D Network diagram showing BRD4 and HMGB2 from the BioGRID database. E Relative protein expression levels detected by western blotting. F Co-IP detection of the BRD4 and HMGB2 combination. G Yeast hybrid system to verify the binding domain of BRD4 (BD1, BD2, ET, and CTD domains) to HMGB2. H Dual-luciferase results showing the effects of BRD4 and HMGB2 over-expression (or down-regulation) on HADH, ACSL1, and ACAA2 promoter activity. ( n = 3, Mean ± SD, one-way ANOVA, * P < 0.05; ** P < 0.01; *** P < 0.005; N.S.: no significant).

Article Snippet: Then, the fragments were precipitated following the kit manufacturer’s instructions using the BRD4 ChIP grade antibody (Abcam, Cat. No. Ab243862) (CST, Cat. No.9003).

Techniques: Co-Immunoprecipitation Assay, Silver Staining, Binding Assay, Expressing, Western Blot, Luciferase, Over Expression, Activity Assay

Journal: Frontiers in Oncology

Article Title: Inhibition of Bromodomain and Extra Terminal (BET) Domain Activity Modulates the IL-23R/IL-17 Axis and Suppresses Acute Graft- Versus -Host Disease

doi: 10.3389/fonc.2021.760789

Figure Lengend Snippet: IC 50 values for PLX51107 and PLX2853 against BET proteins.

Article Snippet: Magnetic beads were pelleted using magnetic stand and the supernatant was incubated with ChIP grade BRD4 antibody (Active Motif) at 4°C overnight with rotation.

Techniques:

Journal: Molecular cell

Article Title: ZFX mediates non-canonical oncogenic functions of the androgen receptor splice variant 7 (AR-V7) in castrate-resistant prostate cancer

doi: 10.1016/j.molcel.2018.08.029

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-BRD4 ChIP Grade , Bethyl , , A301–985A100 , .

Techniques: Immunoprecipitation, Virus, Recombinant, Electron Microscopy, Transfection, Protease Inhibitor, cDNA Synthesis, SYBR Green Assay, Purification, RNA Library Preparation, Multiplex Assay, Proliferation Assay, Mutagenesis, Magnetic Beads, Western Blot, Stripping, Membrane, shRNA, Knockdown, Control, Construct, Plasmid Preparation, Software

Journal: Gynecologic oncology

Article Title: The BET inhibitor INCB054329 reduces homologous recombination efficiency and augments PARP inhibitor activity in ovarian cancer

doi: 10.1016/j.ygyno.2018.03.049

Figure Lengend Snippet: A) TCGA high-grade serous ovarian tumors with BRD2, BRD3 and/or BRD4 amplification overlap with a significant subset of CCNE1 (cyclin E) amplified tumors. Copy number analysis data extracted from www.cbioportal.org. SKOV-3 cells were transiently transfected with distinct non-targeting (NT) or siRNAs targeting BRD4 (siBRD4). After 24 h transfection, the cells were treated with vehicle (0.01% DMSO) or olaparib (10μM) for an additional 24 hours. B) Western blot analysis of expression of BRD4, BRCA1, cleaved PARP and pH2AX. Actin and H2AX were used as loading controls. C) Densitometry of blots from A). Results are expressed relative to corresponding actin or histone H3 levels. Values are mean+SEM of 3 independent experiments; *p<0.01 compared to NT; ap<0.01 relative to olaparib-NT alone, Student’s t-test. D) Chromatin immunoprecipitation strategy for analysis of BRD4 binding to the BRCA1 locus. Primers were designed against a region proximal to the TSS (ChIP-BRCA1) and a negative control region (ChIP-Neg). E) ChIP-QPCR analysis of anti-BRD4 and anti-acetylated histone H3 (AcH3) in OVCAR-3 cells treated with NT or siBRD4 for 48 hours, or vehicle or INCB054329 (1μM) for 6h.

Article Snippet: The sonicated chromatin was immunoprecipitated with 5 μg of rabbit polyclonal ChIP-grade anti-BRD4 antibody (Millipore), 5 μg of a rabbit polyclonal acetylated histone H3 antibody (Millipore), or 5 μg of normal rabbit IgG (Santa Cruz Biotechnology).

Techniques: Amplification, Transfection, Western Blot, Expressing, Chromatin Immunoprecipitation, Binding Assay, Negative Control, ChIP-qPCR

Journal: Gynecologic oncology

Article Title: The BET inhibitor INCB054329 reduces homologous recombination efficiency and augments PARP inhibitor activity in ovarian cancer

doi: 10.1016/j.ygyno.2018.03.049

Figure Lengend Snippet: A) Western blot analysis of expression of BRD4, BRCA1, RAD51 and cyclin E in OVCAR-3 or SKOV-3 cells treated with vehicle (0.01% DMSO), INCB054329 (1μM) and olaparib (10μM) or the combination (24h). Densitometry analysis of blots for B) OVCAR-3 and C) SKOV-3 cells. Results are expressed relative to corresponding actin expression. OVCAR-3 or SKOV-3 cells pre-treated with 0.5μM cisplatin for 6h were then treated as above for a further 24 h. D) Foci for BRCA1 (green) were stained by IF. DAPI stained nuclei are in blue. Representative images are for SKOV-3 cells. E) The percentage of cells positive for BRCA1. F) Foci for RAD51 (green) and pH2AX (red) were stained by IF. Representative images are for SKOV-3 cells. G) The percentage of cells positive for RAD51. Greater than 5 foci per nucleus was considered positive staining for BRCA1 and RAD51. HR repair efficiency of DNA double-strand breaks (DSBs) was also measured using the DRGFP assay. H–I) IF staining for GFP (green) in OVCAR-3 or SKOV3 cells co-transfected with the pDRGFP and I-Sce1 plasmids (both 1μg), and then treated as above for 24h. Representative images are for SKOV-3 cells. Results are expressed as the percentage of GFP-expressing cells. NHEJ repair efficiency was measured using the EJ5GFP assay. J) The percentage of GFP-expressing cells in OVCAR-3 or SKOV3 cells co-transfected with the EJ5GFP and I-Sce1 plasmids (both 1μg), and then treated as above for 24h. K) Drug effects on protein expression of the NHEJ marker, phospho(T2609)-DNA-PKcs and total DNA-PKcs, measured by western blot. For IF assays, at least 100 cells were counted (×40) for each assay and treatment. Values are mean+SEM of 3 independent experiments; *p<0.01 compared to control; ap<0.01 relative to olaparib alone, bp<0.01 relative to INCB054329 alone, Student’s t-test. Scale bars represent 20μm in D) and F), and 40μm in I).

Article Snippet: The sonicated chromatin was immunoprecipitated with 5 μg of rabbit polyclonal ChIP-grade anti-BRD4 antibody (Millipore), 5 μg of a rabbit polyclonal acetylated histone H3 antibody (Millipore), or 5 μg of normal rabbit IgG (Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, Staining, Transfection, Marker, Control