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rabbit monoclonal anti brd4 (Cell Signaling Technology Inc)

Name: rabbit monoclonal anti brd4
Brand: Cell Signaling Technology Inc
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Cell Signaling Technology Inc rabbit monoclonal anti brd4
Rabbit Monoclonal Anti Brd4, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 468 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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O-GlcNAcylation of BRD4 inhibited NF-κB p65-mediated transcription of pro-inflammatory cytokines. (A)&(B) The expression of BRD4 in OGD-exposed cardiomyocytes was detected by RT-qPCR and Western blotting. H9C2 and AC-16 cells were transfected with shBRD4, and then subjected to OGD. (C)&(D) RT-qPCR and Western blotting analysis of BRD4 mRNA and protein levels. (E)&(F) The mRNA levels and concentrations of TNF-α, IL-1β, and IL-6 were determined by RT-qPCR and ELISA. (G) The binding of NF-κB p65 to TNF-α, IL-1β, and IL-6 promoters was confirmed by dual-luciferase reporter assay. (H)&(I) Co-IP assay verified the exogenous and endogenous interplay between OGT and BRD4 proteins. (J) O-GlcNAcylation of BRD4 protein in OGD-stimulated cardiomyocytes was evaluated. (K) YinOYang database predicated the potential O-GlcNAc sites on BRD4. OGD-challenged H9C2 and AC-16 cells were transfected with BRD4 WT plasmid or BRD4 plasmids with mutant O-GlcNAc sites (BRD4-S484R, BRD4-S784R, and BRD4-T1212R). (L) O-GlcNAcylation of BRD4 protein in H9C2 and AC-16 cells was detected. (M) Concentrations of TNF-α, IL-1β, and IL-6 were detected by ELISA. (N) The interaction between NF-κB p65 and TNF-α, IL-1β, and IL-6 promoters was validated by dual-luciferase reporter assay. n=3 for A-N. Student's t test (for A, B) and one-way ANOVA (for C-G, M, N) were performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Theranostics

Article Title: Divergent splicing factor SRSF1 signaling promotes inflammation post-CME: the SRSF1/ENPP3 axis acts via inhibition of BRD4 O-GlcNAcylation to enhance NF-κB activation and accelerate heart failure

doi: 10.7150/thno.115402

Figure Lengend Snippet: O-GlcNAcylation of BRD4 inhibited NF-κB p65-mediated transcription of pro-inflammatory cytokines. (A)&(B) The expression of BRD4 in OGD-exposed cardiomyocytes was detected by RT-qPCR and Western blotting. H9C2 and AC-16 cells were transfected with shBRD4, and then subjected to OGD. (C)&(D) RT-qPCR and Western blotting analysis of BRD4 mRNA and protein levels. (E)&(F) The mRNA levels and concentrations of TNF-α, IL-1β, and IL-6 were determined by RT-qPCR and ELISA. (G) The binding of NF-κB p65 to TNF-α, IL-1β, and IL-6 promoters was confirmed by dual-luciferase reporter assay. (H)&(I) Co-IP assay verified the exogenous and endogenous interplay between OGT and BRD4 proteins. (J) O-GlcNAcylation of BRD4 protein in OGD-stimulated cardiomyocytes was evaluated. (K) YinOYang database predicated the potential O-GlcNAc sites on BRD4. OGD-challenged H9C2 and AC-16 cells were transfected with BRD4 WT plasmid or BRD4 plasmids with mutant O-GlcNAc sites (BRD4-S484R, BRD4-S784R, and BRD4-T1212R). (L) O-GlcNAcylation of BRD4 protein in H9C2 and AC-16 cells was detected. (M) Concentrations of TNF-α, IL-1β, and IL-6 were detected by ELISA. (N) The interaction between NF-κB p65 and TNF-α, IL-1β, and IL-6 promoters was validated by dual-luciferase reporter assay. n=3 for A-N. Student's t test (for A, B) and one-way ANOVA (for C-G, M, N) were performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The sections received overnight incubation with primary antibodies SRSF1 (12929-2-AP, 1:50, Proteintech, Wuhan, China), ENPP3 (A05615, 1:100, Boster, CA, USA), or BRD4 (M00123, 1:50, Boster) at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Binding Assay, Luciferase, Reporter Assay, Co-Immunoprecipitation Assay, Plasmid Preparation, Mutagenesis

ENPP3 contributed to inflammation by inhibiting O-GlcNAcylation of BRD4. H9C2 and AC-16 cells were transfected with shENPP3, followed by exposure to OGD. (A) ENPP3 and BRD4 protein levels were measured by Western blotting. (B) The O-GlcNAc level of BRD4 protein was assessed. (C) The production of TNF-α, IL-1β, and IL-6 was determined by ELISA. (D) Dual-luciferase reporter assay evaluated the binding of NF-κB p65 to TNF-α, IL-1β, and IL-6 promoters. n=3 for A-D. One-way ANOVA was performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Theranostics

doi: 10.7150/thno.115402

Figure Lengend Snippet: ENPP3 contributed to inflammation by inhibiting O-GlcNAcylation of BRD4. H9C2 and AC-16 cells were transfected with shENPP3, followed by exposure to OGD. (A) ENPP3 and BRD4 protein levels were measured by Western blotting. (B) The O-GlcNAc level of BRD4 protein was assessed. (C) The production of TNF-α, IL-1β, and IL-6 was determined by ELISA. (D) Dual-luciferase reporter assay evaluated the binding of NF-κB p65 to TNF-α, IL-1β, and IL-6 promoters. n=3 for A-D. One-way ANOVA was performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, Binding Assay

SRSF1/ENPP3 axis suppressed BRD4 O-GlcNAcylation to promote inflammation in CME. The OGD-stimulated cardiomyocytes were transfected with shSRSF1, ENPP3 overexpression plasmid, or a combination of them. (A) ENPP3 mRNA and lncRNA ENPP3 expression levels were detected by RT-qPCR. (B) The protein abundance of ENPP3 and BRD4 was assessed by Western blotting. (C) The O-GlcNAc level of BRD4 was determined. (D) ELISA was carried out to measure TNF-α, IL-1β, and IL-6 concentrations. n=3 for A-D. One-way ANOVA was performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Theranostics

doi: 10.7150/thno.115402

Figure Lengend Snippet: SRSF1/ENPP3 axis suppressed BRD4 O-GlcNAcylation to promote inflammation in CME. The OGD-stimulated cardiomyocytes were transfected with shSRSF1, ENPP3 overexpression plasmid, or a combination of them. (A) ENPP3 mRNA and lncRNA ENPP3 expression levels were detected by RT-qPCR. (B) The protein abundance of ENPP3 and BRD4 was assessed by Western blotting. (C) The O-GlcNAc level of BRD4 was determined. (D) ELISA was carried out to measure TNF-α, IL-1β, and IL-6 concentrations. n=3 for A-D. One-way ANOVA was performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques: Transfection, Over Expression, Plasmid Preparation, Expressing, Quantitative RT-PCR, Quantitative Proteomics, Western Blot, Enzyme-linked Immunosorbent Assay

Myocardium-specific SRSF1 knockout alleviated CME-induced inflammation via inactivation of the ENPP3/BRD4/NF-κB pathway. SRSF1 flox/flox and SRSF1-KO rats were injected with microspheres into the left ventricle to induce CME. (A) LVEF, LVFS, LVEDd, and CO were detected to evaluate cardiac function. (B) The serum cTnl level in different groups was measured by ELISA. (C) Pathological alterations in myocardial tissues were observed by HE staining (scale bar = 100 μm). (D) Myocardial infarct size was measured by HBFP staining (scale bar = 100 μm). (E) SRSF1, ENPP3, and BRD4 expression in myocardial tissues was evaluated by immunohistochemical staining (scale bar = 100 μm). (F) The protein abundance of SRSF1, ENPP3, BRD4, p65, and O-GlcNAcylation of BRD4 was detected by Western blotting or Co-IP, respectively. (G) ELISA was carried out to measure TNF-α, IL-1β, and IL-6 concentrations. n=6 for A-G. ANOVA for repeated measurement (for A, B), and one-way ANOVA (for F, G) was performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Theranostics

doi: 10.7150/thno.115402

Figure Lengend Snippet: Myocardium-specific SRSF1 knockout alleviated CME-induced inflammation via inactivation of the ENPP3/BRD4/NF-κB pathway. SRSF1 flox/flox and SRSF1-KO rats were injected with microspheres into the left ventricle to induce CME. (A) LVEF, LVFS, LVEDd, and CO were detected to evaluate cardiac function. (B) The serum cTnl level in different groups was measured by ELISA. (C) Pathological alterations in myocardial tissues were observed by HE staining (scale bar = 100 μm). (D) Myocardial infarct size was measured by HBFP staining (scale bar = 100 μm). (E) SRSF1, ENPP3, and BRD4 expression in myocardial tissues was evaluated by immunohistochemical staining (scale bar = 100 μm). (F) The protein abundance of SRSF1, ENPP3, BRD4, p65, and O-GlcNAcylation of BRD4 was detected by Western blotting or Co-IP, respectively. (G) ELISA was carried out to measure TNF-α, IL-1β, and IL-6 concentrations. n=6 for A-G. ANOVA for repeated measurement (for A, B), and one-way ANOVA (for F, G) was performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques: Knock-Out, Injection, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Immunohistochemical staining, Quantitative Proteomics, Western Blot, Co-Immunoprecipitation Assay

Figure 1. JNK directly interacts with and phosphorylates BRD4 (A) BRD4 co-localizes with kinase active JNK. Proximity ligation assays (PLAs) with anti-BRD4 and anti-pJNK on ﬁxed HCT116 cells. Negative control; anti- nucleolin, and anti-BRD4 (scale bars, 20 mM). (B) JNK co-immunoprecipitates with BRD4. BRD4 was immunoprecipitated from HeLa nuclear extract using anti-BRD4 and immunoblotted with anti-JNK. (C) BRD4 binds JNK directly. Recombinant JNK1 (0.1 and 0.2 mg) was pulled down with 0.5 mg rBRD4 immobilized on FLAG beads. (D) JNK phosphorylates BRD4. Upper: map of BRD4 and deletion mutants. Lower: autoradiograph of kinase assays with GST-JNK1 and WT-BRD4 or deletion mutants. (E) JNK phosphorylation sites on BRD4. Upper: JNK consensus phosphorylation sites located on human/mouse BRD4. Lower: autoradiograph of kinase assays with His-JNK1 and BRD4 WT or the point mutants. (F) BRD4 is phosphorylated at Thr1186 and Thr1212 JNK activation. HCT116 cells were treated with anisomycin, heat shock, LPS treatment, or UV stress. BRD4 phosphorylation was assessed by immunoblotting (upper) and densitometric quantiﬁcation (lower). (G) BRD4’s interaction with JNK is abrogated by phosphorylation. CoIP of JNK with BRD4 following anisomycin treatment of WT- and 3A-BRD4-expressing HCT116 cells. See also Figures S1 and S2.

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 1. JNK directly interacts with and phosphorylates BRD4 (A) BRD4 co-localizes with kinase active JNK. Proximity ligation assays (PLAs) with anti-BRD4 and anti-pJNK on ﬁxed HCT116 cells. Negative control; anti- nucleolin, and anti-BRD4 (scale bars, 20 mM). (B) JNK co-immunoprecipitates with BRD4. BRD4 was immunoprecipitated from HeLa nuclear extract using anti-BRD4 and immunoblotted with anti-JNK. (C) BRD4 binds JNK directly. Recombinant JNK1 (0.1 and 0.2 mg) was pulled down with 0.5 mg rBRD4 immobilized on FLAG beads. (D) JNK phosphorylates BRD4. Upper: map of BRD4 and deletion mutants. Lower: autoradiograph of kinase assays with GST-JNK1 and WT-BRD4 or deletion mutants. (E) JNK phosphorylation sites on BRD4. Upper: JNK consensus phosphorylation sites located on human/mouse BRD4. Lower: autoradiograph of kinase assays with His-JNK1 and BRD4 WT or the point mutants. (F) BRD4 is phosphorylated at Thr1186 and Thr1212 JNK activation. HCT116 cells were treated with anisomycin, heat shock, LPS treatment, or UV stress. BRD4 phosphorylation was assessed by immunoblotting (upper) and densitometric quantiﬁcation (lower). (G) BRD4’s interaction with JNK is abrogated by phosphorylation. CoIP of JNK with BRD4 following anisomycin treatment of WT- and 3A-BRD4-expressing HCT116 cells. See also Figures S1 and S2.

Article Snippet: The primary antibodies used were as follows: anti-BRD4 rabbit monoclonal antibody (Bethyl; [BL-149-2H5]) (1:100 dilution), anti-phospho JNK mouse monoclonal antibody (G-7, Santa cruz biotechnology) (1:100 dilution), and anti-Nucleolin (sc-8031, Santa cruz biotechnology) (1:100 dilution).

Techniques: Ligation, Negative Control, Immunoprecipitation, Recombinant, Autoradiography, Phospho-proteomics, Activation Assay, Western Blot, Expressing

Figure 2. JNK phosphorylation of BRD4 releases it from chromatin (A) BRD4 is released from the chromatin upon JNK activation by anisomycin. Immunoblots of chromatin-free (CF) and chromatin-bound (CB) BRD4 in HCT116 cells following treatment with anisomycin. (B) Inhibition of JNK kinase blocks BRD4’s release from chromatin. Immunoblots of CF and CB BRD4 in HCT116 cells transfected with WT JNK1 or JNK1 and dominant-negative kinase mutants JNK1/JNK2 (APF) individually or in combination, followed by anisomycin treatment. (C) BRD4 is released from the chromatin upon JNK activation by heat shock. Left: immunoblots of CF and CB BRD4 in HCT116 cells grown at 37C or heat shocked at 42C for 15 min in the presence or absence of JNK inhibitor SP600125. Right: immunoblots showing pJNK levels under the above conditions. (D) JNK preferentially phosphorylates BRD4 bound to mononucleosomes. Anti-BRD4 pT1212 and anti-BRD4 immunoblots of kinase assays with recombinant JNK1 and BRD4 after pre-incubating BRD4 with or without assembled mononucleosomes (MN) for 10 or 20 min. (E) Mutation of BRD4 phosphorylation sites prevents BRD4 release from chromatin. Left: immunoblots of CF and CB BRD4 in HCT116 cells transfected with WT or 3A-BRD4 and subjected to heat shock treatment. Right: immunoblots showing pJNK levels following heat shock in WT- and 3A-BRD4-expressing cells. (F) JNK activation results in global loss of CB BRD4. Total BRD4 peaks detected in BRD4 ChIP-seq of control-untreated and anisomycin-treated DLD1 BRD4- IAA7 cells expressing endogenous BRD4 or exogenous WT or 3A-BRD4 following auxin treatment. (G) Loss of JNK-phosphorylated BRD4 from chromatin is widespread. Distribution of BRD4 ChIP-seq peaks across the genomes of cells described in (F).

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 2. JNK phosphorylation of BRD4 releases it from chromatin (A) BRD4 is released from the chromatin upon JNK activation by anisomycin. Immunoblots of chromatin-free (CF) and chromatin-bound (CB) BRD4 in HCT116 cells following treatment with anisomycin. (B) Inhibition of JNK kinase blocks BRD4’s release from chromatin. Immunoblots of CF and CB BRD4 in HCT116 cells transfected with WT JNK1 or JNK1 and dominant-negative kinase mutants JNK1/JNK2 (APF) individually or in combination, followed by anisomycin treatment. (C) BRD4 is released from the chromatin upon JNK activation by heat shock. Left: immunoblots of CF and CB BRD4 in HCT116 cells grown at 37C or heat shocked at 42C for 15 min in the presence or absence of JNK inhibitor SP600125. Right: immunoblots showing pJNK levels under the above conditions. (D) JNK preferentially phosphorylates BRD4 bound to mononucleosomes. Anti-BRD4 pT1212 and anti-BRD4 immunoblots of kinase assays with recombinant JNK1 and BRD4 after pre-incubating BRD4 with or without assembled mononucleosomes (MN) for 10 or 20 min. (E) Mutation of BRD4 phosphorylation sites prevents BRD4 release from chromatin. Left: immunoblots of CF and CB BRD4 in HCT116 cells transfected with WT or 3A-BRD4 and subjected to heat shock treatment. Right: immunoblots showing pJNK levels following heat shock in WT- and 3A-BRD4-expressing cells. (F) JNK activation results in global loss of CB BRD4. Total BRD4 peaks detected in BRD4 ChIP-seq of control-untreated and anisomycin-treated DLD1 BRD4- IAA7 cells expressing endogenous BRD4 or exogenous WT or 3A-BRD4 following auxin treatment. (G) Loss of JNK-phosphorylated BRD4 from chromatin is widespread. Distribution of BRD4 ChIP-seq peaks across the genomes of cells described in (F).

Techniques: Phospho-proteomics, Activation Assay, Western Blot, Inhibition, Transfection, Dominant Negative Mutation, Recombinant, Mutagenesis, Expressing, ChIP-sequencing, Control

Figure 3. JNK-mediated BRD4 release from chromatin disrupts its nucleosome clearance function (A) BRD4 binding to mononucleosomes is abrogated upon phosphorylation by JNK. Anti-histone H3 immunoblot of assembled mononucleosomes pulled down by recombinant WT or 3A-BRD4 that was either unphosphorylated or pre-phosphorylated (*) by JNK and immobilized on FLAG beads. (B) JNK phosphorylation of BRD4 inhibits H3K122 acetylation. Anti-histone H3K122ac immunoblot of assembled mononucleosomes subjected to an in vitro HAT assay with recombinant WT or 3A-BRD4 that was either unphosphorylated or pre-phosphorylated (*) by JNK. (C) H3K122 acetylation is reduced in vivo upon JNK activation. Immunoblots of whole-cell extracts (WCEs) of HCT116 cells that were untreated (control) or treated with either DMSO (mock) or anisomycin. (D) In vivo H3K122 acetylation by BRD4 is regulated by JNK. Immunoblots of WCEs of HCT116 cells that were transfected with WT or 3A-BRD4 and treated with or without anisomycin. (E) In vivo H3K122 acetylation is regulated by JNK kinase activity. Immunoblots of WCEs of HCT116 cells that were transfected with WT JNK1, JNK2, or their respective dominant-negative mutants (JNK APF), either individually or together, and treated with or without anisomycin. Densitometric quantiﬁcation of H3K122ac levels is shown below. (F) Nucleosome clearance activity by BRD4 is controlled by JNK phosphorylation. Autoradiograph of an in vitro nucleosome clearance assay showing disso- ciation of assembled mononucleosomes by unphosphorylated or JNK pre-phosphorylated (*) WT or 3A-BRD4 upon being subjected to a HAT assay in the presence or absence of AcCoA. See also Figure S4.

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 3. JNK-mediated BRD4 release from chromatin disrupts its nucleosome clearance function (A) BRD4 binding to mononucleosomes is abrogated upon phosphorylation by JNK. Anti-histone H3 immunoblot of assembled mononucleosomes pulled down by recombinant WT or 3A-BRD4 that was either unphosphorylated or pre-phosphorylated (*) by JNK and immobilized on FLAG beads. (B) JNK phosphorylation of BRD4 inhibits H3K122 acetylation. Anti-histone H3K122ac immunoblot of assembled mononucleosomes subjected to an in vitro HAT assay with recombinant WT or 3A-BRD4 that was either unphosphorylated or pre-phosphorylated (*) by JNK. (C) H3K122 acetylation is reduced in vivo upon JNK activation. Immunoblots of whole-cell extracts (WCEs) of HCT116 cells that were untreated (control) or treated with either DMSO (mock) or anisomycin. (D) In vivo H3K122 acetylation by BRD4 is regulated by JNK. Immunoblots of WCEs of HCT116 cells that were transfected with WT or 3A-BRD4 and treated with or without anisomycin. (E) In vivo H3K122 acetylation is regulated by JNK kinase activity. Immunoblots of WCEs of HCT116 cells that were transfected with WT JNK1, JNK2, or their respective dominant-negative mutants (JNK APF), either individually or together, and treated with or without anisomycin. Densitometric quantiﬁcation of H3K122ac levels is shown below. (F) Nucleosome clearance activity by BRD4 is controlled by JNK phosphorylation. Autoradiograph of an in vitro nucleosome clearance assay showing disso- ciation of assembled mononucleosomes by unphosphorylated or JNK pre-phosphorylated (*) WT or 3A-BRD4 upon being subjected to a HAT assay in the presence or absence of AcCoA. See also Figure S4.

Techniques: Binding Assay, Phospho-proteomics, Western Blot, Recombinant, In Vitro, HAT Assay, In Vivo, Activation Assay, Control, Transfection, Activity Assay, Dominant Negative Mutation, Autoradiography

Figure 4. JNK-mediated BRD4 release from chromatin activates BRD4 kinase (A) JNK activation induces phosphorylation of BRD4 kinase substrates. Immunoblots of WCEs of HCT116 cells that were treated with DMSO (mock), anisomycin, or anisomycin with JNK peptide inhibitor D-JNK1. (B) Blocking JNK activity inhibits induction of BRD4 kinase. Immunoblots of WCEs of HCT116 cells that were transfected with FLAG-tagged WT JNK1, JNK2, or respective dominant-negative mutants (JNK APF), either individually or together, and treated with or without anisomycin. (C) JNK phosphorylation of BRD4 is necessary for induction of BRD4 kinase. Immunoblots of WCEs of HCT116 cells transfected with WT or 3A-BRD4 and subjected to heat shock. (D) BRD4 phosphorylation regulates Myc stability. Immunoﬂuorescence images showing Myc levels in HCT116 cells transfected with either WT or 3A-BRD4 or empty vector (control) and subjected to heat shock (scale bars, 25 mM). See also Figure S5.

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 4. JNK-mediated BRD4 release from chromatin activates BRD4 kinase (A) JNK activation induces phosphorylation of BRD4 kinase substrates. Immunoblots of WCEs of HCT116 cells that were treated with DMSO (mock), anisomycin, or anisomycin with JNK peptide inhibitor D-JNK1. (B) Blocking JNK activity inhibits induction of BRD4 kinase. Immunoblots of WCEs of HCT116 cells that were transfected with FLAG-tagged WT JNK1, JNK2, or respective dominant-negative mutants (JNK APF), either individually or together, and treated with or without anisomycin. (C) JNK phosphorylation of BRD4 is necessary for induction of BRD4 kinase. Immunoblots of WCEs of HCT116 cells transfected with WT or 3A-BRD4 and subjected to heat shock. (D) BRD4 phosphorylation regulates Myc stability. Immunoﬂuorescence images showing Myc levels in HCT116 cells transfected with either WT or 3A-BRD4 or empty vector (control) and subjected to heat shock (scale bars, 25 mM). See also Figure S5.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Blocking Assay, Activity Assay, Transfection, Dominant Negative Mutation, Plasmid Preparation, Control

Figure 5. JNK phosphorylation toggles BRD4 enzymatic activities and is reversed by PP4 phosphatase (A) BRD4 kinase and HAT activities are cross-regulated by its substrates. Top: autoradiograph of an in vitro kinase assay with BRD4 and RNA Pol II CTD in the presence or absence of assembled mononucleosomes. Bottom: immunoblots of an in vitro HAT assay with BRD4 and histone H3 in the presence or absence of RNA Pol II CTD. (B) JNK activation enhances the interaction between BRD4 and its kinase substrates. Immunoblots showing co-immunoprecipitated total and T1212-phos- phorylated BRD4 from HCT116 cells treated with or without anisomycin. Top: BRD4 co-immunoprecipitated with RNA Pol II CTD. Bottom: BRD4 co-immu- noprecipitated with CDK9. (C) JNK-mediated phosphorylation of BRD4 is transient. Immunoblots of WCEs of HCT116 cells grown under normal conditions, subjected to heat shock or heat shocked and then rescued for 20 min. (D) Inhibition of phosphatases enhances phosphorylated BRD4 levels. Immunoblots of WCEs of HCT116 cells that were treated with or without anisomycin alone or anisomycin with phosphatase inhibitor, nodularin. (E) Phosphatase PP4 dephosphorylates JNK-phosphorylated BRD4. Immunoblots of WCEs of HCT116 cells that were transfected with either control, PP2Ac, or PP4c siRNA and treated with or without anisomycin. (F) BRD4’s interaction with RNA Pol II CTD is modulated by PP4. Immunoblots showing total and pT1212 BRD4 co-immunoprecipitated with RNA Pol II CTD from HCT116 cells transfected with either control or PP4c siRNA and treated with anisomycin.

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 5. JNK phosphorylation toggles BRD4 enzymatic activities and is reversed by PP4 phosphatase (A) BRD4 kinase and HAT activities are cross-regulated by its substrates. Top: autoradiograph of an in vitro kinase assay with BRD4 and RNA Pol II CTD in the presence or absence of assembled mononucleosomes. Bottom: immunoblots of an in vitro HAT assay with BRD4 and histone H3 in the presence or absence of RNA Pol II CTD. (B) JNK activation enhances the interaction between BRD4 and its kinase substrates. Immunoblots showing co-immunoprecipitated total and T1212-phos- phorylated BRD4 from HCT116 cells treated with or without anisomycin. Top: BRD4 co-immunoprecipitated with RNA Pol II CTD. Bottom: BRD4 co-immu- noprecipitated with CDK9. (C) JNK-mediated phosphorylation of BRD4 is transient. Immunoblots of WCEs of HCT116 cells grown under normal conditions, subjected to heat shock or heat shocked and then rescued for 20 min. (D) Inhibition of phosphatases enhances phosphorylated BRD4 levels. Immunoblots of WCEs of HCT116 cells that were treated with or without anisomycin alone or anisomycin with phosphatase inhibitor, nodularin. (E) Phosphatase PP4 dephosphorylates JNK-phosphorylated BRD4. Immunoblots of WCEs of HCT116 cells that were transfected with either control, PP2Ac, or PP4c siRNA and treated with or without anisomycin. (F) BRD4’s interaction with RNA Pol II CTD is modulated by PP4. Immunoblots showing total and pT1212 BRD4 co-immunoprecipitated with RNA Pol II CTD from HCT116 cells transfected with either control or PP4c siRNA and treated with anisomycin.

Techniques: Phospho-proteomics, Autoradiography, In Vitro, Kinase Assay, Western Blot, HAT Assay, Activation Assay, Immunoprecipitation, Inhibition, Transfection, Control

Figure 6. JNK-mediated BRD4 release from chromatin activates transcription (A) JNK activation enhances expression of BRD4-regulated genes. Volcano plots showing differential gene expression observed in RNA-seq analysis of WT- and 3A-BRD4-expressing HCT116 cells after anisomycin treatment. (B) Inﬂammatory and immune response pathways are enriched among the BRD4-regulated genes induced by JNK activation. GO analysis of genes induced in anisomycin-treated WT-BRD4-expressing cells relative to control HCT116 cells. (C) Induction of key inﬂammatory and immune response genes depends on JNK phosphorylation of BRD4. RT-qPCR of cDNA from anisomycin-treated WT- and 3A-BRD4-expressing cells relative to control cells. Error bars, SEM (n = 3 independent experiments; *p < 0.001 by two-tailed Student’s t tests). (D) JNK activation leads to increased BRD4-RNA Pol II interaction at BRD4-regulated inﬂammatory and immune response genes. Sequential-ChIP assays showing RNA Pol II and RNA Pol II-bound BRD4 at the promoter and gene body regions of CCL20, CXCL1, BIRC3, and control Myc genes. Error bars, SEM (n = 4 technical replicates from 2 independent experiments; *p < 0.05 by two-tailed Student’s t tests). See also Figure S6.

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 6. JNK-mediated BRD4 release from chromatin activates transcription (A) JNK activation enhances expression of BRD4-regulated genes. Volcano plots showing differential gene expression observed in RNA-seq analysis of WT- and 3A-BRD4-expressing HCT116 cells after anisomycin treatment. (B) Inﬂammatory and immune response pathways are enriched among the BRD4-regulated genes induced by JNK activation. GO analysis of genes induced in anisomycin-treated WT-BRD4-expressing cells relative to control HCT116 cells. (C) Induction of key inﬂammatory and immune response genes depends on JNK phosphorylation of BRD4. RT-qPCR of cDNA from anisomycin-treated WT- and 3A-BRD4-expressing cells relative to control cells. Error bars, SEM (n = 3 independent experiments; *p < 0.001 by two-tailed Student’s t tests). (D) JNK activation leads to increased BRD4-RNA Pol II interaction at BRD4-regulated inﬂammatory and immune response genes. Sequential-ChIP assays showing RNA Pol II and RNA Pol II-bound BRD4 at the promoter and gene body regions of CCL20, CXCL1, BIRC3, and control Myc genes. Error bars, SEM (n = 4 technical replicates from 2 independent experiments; *p < 0.05 by two-tailed Student’s t tests). See also Figure S6.

Techniques: Activation Assay, Expressing, Gene Expression, RNA Sequencing, Control, Phospho-proteomics, Quantitative RT-PCR, Two Tailed Test

Figure 7. BRD4 phosphorylation and chromatin release are correlated with thymocyte activation and EMT (A) Thymocyte activation correlates with JNK phosphorylation of BRD4. Left: ﬂow cytometry proﬁles of thymocytes activated by 0.3 ng PMA/0.3 mg ionomycin or by 10 ng PMA/3.75 mg ionomycin. FACS analysis of CD4/CD8 (upper) and CD69 expression (lower). Right: immunoblots of WCEs from unstimulated and stimulated thymocytes. Densitometric quantiﬁcation of relative BRD4 phosphorylation levels is shown below. (B) Thymocyte activation is correlated with JNK-mediated release of BRD4 from chromatin. Immunoblot of chromatin-free (CF) and chromatin-bound (CB) BRD4 in thymocytes unstimulated or stimulated as described above. Densitometric quantiﬁcation of CF:CB BRD4 ratio is shown below. Anti-histone H3 immunoblot monitors purity of separation. (C) Immunoblots of WCEs from PC3 cells at day 0 and day 5 of treatment with or without EMT-inducing media supplement. (D) EMT induction correlates with JNK-mediated release of BRD4 from chromatin. Immunoblot of CF and CB BRD4 in PC3 cells after 5 days of treatment with or without (control) EMT-inducing media. (E) EMT induction and BRD4 phosphorylation are both dependent on JNK activity. Immunoblots of WCEs from PC3 cells that were treated, or not, with EMT- inducing media alone or in combination with JNK peptide inhibitor D-JNK-1. (F) EMT induction and expression of EMT regulators are dependent on BRD4 phosphorylation. Immunoblots of WCEs from PC3 cells that were treated, or not, with EMT-inducing media and transfected with WT-BRD4, 3A-BRD4, or empty vector control on day 3 of treatment.

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 7. BRD4 phosphorylation and chromatin release are correlated with thymocyte activation and EMT (A) Thymocyte activation correlates with JNK phosphorylation of BRD4. Left: ﬂow cytometry proﬁles of thymocytes activated by 0.3 ng PMA/0.3 mg ionomycin or by 10 ng PMA/3.75 mg ionomycin. FACS analysis of CD4/CD8 (upper) and CD69 expression (lower). Right: immunoblots of WCEs from unstimulated and stimulated thymocytes. Densitometric quantiﬁcation of relative BRD4 phosphorylation levels is shown below. (B) Thymocyte activation is correlated with JNK-mediated release of BRD4 from chromatin. Immunoblot of chromatin-free (CF) and chromatin-bound (CB) BRD4 in thymocytes unstimulated or stimulated as described above. Densitometric quantiﬁcation of CF:CB BRD4 ratio is shown below. Anti-histone H3 immunoblot monitors purity of separation. (C) Immunoblots of WCEs from PC3 cells at day 0 and day 5 of treatment with or without EMT-inducing media supplement. (D) EMT induction correlates with JNK-mediated release of BRD4 from chromatin. Immunoblot of CF and CB BRD4 in PC3 cells after 5 days of treatment with or without (control) EMT-inducing media. (E) EMT induction and BRD4 phosphorylation are both dependent on JNK activity. Immunoblots of WCEs from PC3 cells that were treated, or not, with EMT- inducing media alone or in combination with JNK peptide inhibitor D-JNK-1. (F) EMT induction and expression of EMT regulators are dependent on BRD4 phosphorylation. Immunoblots of WCEs from PC3 cells that were treated, or not, with EMT-inducing media and transfected with WT-BRD4, 3A-BRD4, or empty vector control on day 3 of treatment.

Techniques: Phospho-proteomics, Activation Assay, Cytometry, Expressing, Western Blot, Control, Activity Assay, Transfection, Plasmid Preparation

Figure 8. Model of BRD4-JNK interaction and the switching of BRD4 functions BRD4 primarily functions as a chromatin regulator by acetylating H3K122 and dissociating nucleosomes. Upon activation, JNK phosphorylates BRD4, releasing it from chromatin and activating its kinase. Chromatin-free BRD4 is then dephosphorylated by PP4, enhancing its interaction with and phosphorylation of RNA Pol II CTD, PTEFb, and Myc, thereby activating transcription at speciﬁc genes. A portion of dephosphorylated BRD4 returns to chromatin to renew its chromatin regulatory function.

Journal: Molecular cell

Article Title: Phosphorylation by JNK switches BRD4 functions.

doi: 10.1016/j.molcel.2024.09.030

Figure Lengend Snippet: Figure 8. Model of BRD4-JNK interaction and the switching of BRD4 functions BRD4 primarily functions as a chromatin regulator by acetylating H3K122 and dissociating nucleosomes. Upon activation, JNK phosphorylates BRD4, releasing it from chromatin and activating its kinase. Chromatin-free BRD4 is then dephosphorylated by PP4, enhancing its interaction with and phosphorylation of RNA Pol II CTD, PTEFb, and Myc, thereby activating transcription at speciﬁc genes. A portion of dephosphorylated BRD4 returns to chromatin to renew its chromatin regulatory function.

Techniques: Activation Assay, Phospho-proteomics

Cdk12 ablation increases AR- and MYC-mediated signaling and promotes TRCs (A) Protein expression of CDK12, AR, and FOXA1 in multiple monoclonal Cdk12 WT and Cdk12 KO organoid lines. (GAPDH, loading control). (B) Gene set enrichment of AR target genes (activated and repressed) in Cdk12 KO organoids compared to Cdk12 WT . (C) Proliferation of Cdk12 WT and Cdk12 KO organoids grown in the absence of epidermal growth factor (EGF) and dihydrotestosterone (DHT) as measured by the CTG assay. ( n = 3 replicates per group in 2 unique experiments). (D and E) Morphology and viability quantification of Cdk12 WT and Cdk12 KO organoids subjected to enzalutamide (Enza) treatment. ( n = 3 replicates per group in 2 unique experiments). ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, not significant. (F) Protein expression of CDK12, MYC, BRD4, BRD3, and BRD2 in Cdk12 WT and Cdk12 KO organoid lines. (G) Gene set enrichment of MYC target genes in Cdk12 KO organoids compared to Cdk12 WT . (H) Morphology of Cdk12 WT and Cdk12 KO organoid lines treated with JQ1 (1 μM). ( n = 3/group in 2 unique experiments). (I) Viability curves and IC 50 values for JQ1-treated Cdk12 WT and Cdk12 KO organoid lines. (J) Dot blot analysis quantifying R-loops in Cdk12 WT and Cdk12 KO organoids. RNase H1 treatment serves as a negative control. (K) Immunofluorescence images of R-loop (red) staining of Cdk12 WT and Cdk12 KO organoids (left) and quantification of fluorescence intensity (right). 100–200 cells/group. (L) Experimental workflow for identification of TRCs. Briefly, 2.5 mM of Thymidine was used to synchronize the cells, and 75 μM of DRB was used to inhibit transcription. (M) Representative immunofluorescence images of γH2AX staining in organoids treated as described in (L). (N) Quantification of γH2AX-positive cells in (M); ( n = 6/group, 3 unique experiments conducted). (O) Representative immunofluorescence images of γH2AX staining in unsynchronized organoids. (P) Quantification of γH2AX-positive cells in (O); n = 6–8 per group (3 unique experiments conducted). (Q) Detection of TRC by PLA assay. (R) Quantification of PLA foci per nucleus in (Q); 100–400 cells analyzed per group (2 unique experiments conducted). Data represented as mean ± SEM. One-way ANOVA for multiple comparisons, two-way ANOVA for multiple variables.

Journal: Cell Reports Medicine

Article Title: CDK12 loss drives prostate cancer progression, transcription-replication conflicts, and synthetic lethality with paralog CDK13

doi: 10.1016/j.xcrm.2024.101758

Figure Lengend Snippet: Cdk12 ablation increases AR- and MYC-mediated signaling and promotes TRCs (A) Protein expression of CDK12, AR, and FOXA1 in multiple monoclonal Cdk12 WT and Cdk12 KO organoid lines. (GAPDH, loading control). (B) Gene set enrichment of AR target genes (activated and repressed) in Cdk12 KO organoids compared to Cdk12 WT . (C) Proliferation of Cdk12 WT and Cdk12 KO organoids grown in the absence of epidermal growth factor (EGF) and dihydrotestosterone (DHT) as measured by the CTG assay. ( n = 3 replicates per group in 2 unique experiments). (D and E) Morphology and viability quantification of Cdk12 WT and Cdk12 KO organoids subjected to enzalutamide (Enza) treatment. ( n = 3 replicates per group in 2 unique experiments). ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, not significant. (F) Protein expression of CDK12, MYC, BRD4, BRD3, and BRD2 in Cdk12 WT and Cdk12 KO organoid lines. (G) Gene set enrichment of MYC target genes in Cdk12 KO organoids compared to Cdk12 WT . (H) Morphology of Cdk12 WT and Cdk12 KO organoid lines treated with JQ1 (1 μM). ( n = 3/group in 2 unique experiments). (I) Viability curves and IC 50 values for JQ1-treated Cdk12 WT and Cdk12 KO organoid lines. (J) Dot blot analysis quantifying R-loops in Cdk12 WT and Cdk12 KO organoids. RNase H1 treatment serves as a negative control. (K) Immunofluorescence images of R-loop (red) staining of Cdk12 WT and Cdk12 KO organoids (left) and quantification of fluorescence intensity (right). 100–200 cells/group. (L) Experimental workflow for identification of TRCs. Briefly, 2.5 mM of Thymidine was used to synchronize the cells, and 75 μM of DRB was used to inhibit transcription. (M) Representative immunofluorescence images of γH2AX staining in organoids treated as described in (L). (N) Quantification of γH2AX-positive cells in (M); ( n = 6/group, 3 unique experiments conducted). (O) Representative immunofluorescence images of γH2AX staining in unsynchronized organoids. (P) Quantification of γH2AX-positive cells in (O); n = 6–8 per group (3 unique experiments conducted). (Q) Detection of TRC by PLA assay. (R) Quantification of PLA foci per nucleus in (Q); 100–400 cells analyzed per group (2 unique experiments conducted). Data represented as mean ± SEM. One-way ANOVA for multiple comparisons, two-way ANOVA for multiple variables.

Article Snippet: Rabbit monoclonal anti-BRD4 antibody , Bethyl , Cat#A700-004; RRID: AB_2631885.

Techniques: Expressing, Control, CTG Assay, Dot Blot, Negative Control, Immunofluorescence, Staining, Fluorescence

Journal: Cell Reports Medicine

Article Title: CDK12 loss drives prostate cancer progression, transcription-replication conflicts, and synthetic lethality with paralog CDK13

doi: 10.1016/j.xcrm.2024.101758

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-BRD4 antibody , Bethyl , Cat#A700-004; RRID: AB_2631885.

Techniques: Virus, Recombinant, Plasmid Preparation, Blocking Assay, Lysis, Protease Inhibitor, Membrane, Transfection, SYBR Green Assay, Cell Viability Assay, Avidin-Biotin Assay, cDNA Synthesis, Software

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