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anti brd9 (Bethyl)

Name: anti brd9
Brand: Bethyl
Rating: 94 (55 reviews)

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Bethyl anti brd9
Anti Brd9, supplied by Bethyl, used in various techniques. Bioz Stars score: 94/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti brd9/product/Bethyl
Average 94 stars, based on 55 article reviews

anti brd9 - by Bioz Stars, 2026-03

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(A) Schematic showing how the Parbit expression cassette is used to generate stably expressed Acyl-eCRs in mESCs. RMCE by Cre, followed by a double selection of ganciclovir and puromycin, was applied to generate these constructs at a defined site in the mouse genome. A CAG promoter drives the constitutive expression of the bromodomain of interest, which is fused to a nuclear localization signal (NLS), and an eGFP tag. This construct also fuses a biotin acceptor site to the N-terminus of the protein, which can be biotinylated in vivo by a bacterial BirA ligase. (B) Schematic diagram showing how CBP was endogenously tagged with an eGFP tag. A homology donor construct was generated by cloning a 900 bp upstream and a 1,048 bp (CBP) or 1,068 bp (p300) downstream homology arm flanking a 30 bp flexible GGS linker that was fused to an eGFP tag. This donor construct was co-transfected with a pX330 CRISPR-Cas9 plasmid, which had an sgRNA targeting the C-terminus of the CBP gene. (C-D) Sanger sequencing of genotyping PCR products from the C-terminus of the CBP (C) and p300 (D) loci, confirming the in-frame homologous integration of an eGFP tag. Data shown are from mESC clone #1 for both CBP and p300 tagging. (E) Flow cytometry data showing the eGFP signal from cell lines where either CBP or p300 were endogenously tagged with eGFP. Two clonal replicates for each protein tagging are shown. Cell lines were maintained in culture for more than two weeks to demonstrate stable expression of the eGFP fusion proteins. (F) Western blot of nuclear extracts from cell lines treated with 1 μM dCBP-1 PROTAC for the stated duration. An antibody against GFP was used to probe the eGFP tag on the Acyl-eCR constructs. The CBP_BRD.1x eCR runs at approximately 60 kDa, and the Empty-eGFP construct runs at 33 kDa. Non-specific bands are marked by an asterisk (*). Revert700 Total Protein Stain is used to show equal loading in lanes.

Journal: bioRxiv

Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

doi: 10.1101/2025.09.06.674632

Figure Lengend Snippet: (A) Schematic showing how the Parbit expression cassette is used to generate stably expressed Acyl-eCRs in mESCs. RMCE by Cre, followed by a double selection of ganciclovir and puromycin, was applied to generate these constructs at a defined site in the mouse genome. A CAG promoter drives the constitutive expression of the bromodomain of interest, which is fused to a nuclear localization signal (NLS), and an eGFP tag. This construct also fuses a biotin acceptor site to the N-terminus of the protein, which can be biotinylated in vivo by a bacterial BirA ligase. (B) Schematic diagram showing how CBP was endogenously tagged with an eGFP tag. A homology donor construct was generated by cloning a 900 bp upstream and a 1,048 bp (CBP) or 1,068 bp (p300) downstream homology arm flanking a 30 bp flexible GGS linker that was fused to an eGFP tag. This donor construct was co-transfected with a pX330 CRISPR-Cas9 plasmid, which had an sgRNA targeting the C-terminus of the CBP gene. (C-D) Sanger sequencing of genotyping PCR products from the C-terminus of the CBP (C) and p300 (D) loci, confirming the in-frame homologous integration of an eGFP tag. Data shown are from mESC clone #1 for both CBP and p300 tagging. (E) Flow cytometry data showing the eGFP signal from cell lines where either CBP or p300 were endogenously tagged with eGFP. Two clonal replicates for each protein tagging are shown. Cell lines were maintained in culture for more than two weeks to demonstrate stable expression of the eGFP fusion proteins. (F) Western blot of nuclear extracts from cell lines treated with 1 μM dCBP-1 PROTAC for the stated duration. An antibody against GFP was used to probe the eGFP tag on the Acyl-eCR constructs. The CBP_BRD.1x eCR runs at approximately 60 kDa, and the Empty-eGFP construct runs at 33 kDa. Non-specific bands are marked by an asterisk (*). Revert700 Total Protein Stain is used to show equal loading in lanes.

Article Snippet: The CBP/p300 bromodomain inhibitor: GNE-049 (MedChemExpress, HY-108435), CBP/p300 PROTAC: dCBP-1 (MedChemExpress, HY-134582), BRD4 bromodomain inhibitor: (+)-JQ-1 (MedChemExpress, HY-13030), BRD4 PROTAC: ARV-825 (MedChemExpress, HY-16954), BRD9 bromodomain inhibitor: iBRD9 (MedChemExpress, HY-18975), and broad-spectrum bromodomain inhibitor: Bromosporine (MedChemExpress, HY-15815) were dissolved in DMSO and then diluted to 1μM in mESC media for 24-hour treatments, unless stated otherwise.

Techniques: Expressing, Stable Transfection, Selection, Construct, In Vivo, Generated, Cloning, Transfection, CRISPR, Plasmid Preparation, Sequencing, Flow Cytometry, Western Blot, Staining

(A) Schematic showing the domain architecture of the Brd4 protein, and how its bromodomains are being used in several combinations to make acyl-eCRs and to determine how the valency of reader domains affects drug perturbations. (B) Immunofluorescence images of mESCs showing the nuclear localization of different valencies of the second bromodomain from BRD4 in the Parbit system (green) and their colocalization with Hoechst (magenta) after drug treatments. All scale bars are 5 µM. Drug treatments were performed at 1 μM concentrations for 24 hours. Bottom panel: Representative pseudocolored images (eGFP signal) depicting the differences in fluorescence intensities in different cell lines. A gradient pseudocolor bar (signal intensity) is shown at the left. (C) Normalized FACS data showing the effects of ARV-825 PROTAC treatment on cells expressing several combinations of bromodomains from BRD4. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

Journal: bioRxiv

Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

doi: 10.1101/2025.09.06.674632

Figure Lengend Snippet: (A) Schematic showing the domain architecture of the Brd4 protein, and how its bromodomains are being used in several combinations to make acyl-eCRs and to determine how the valency of reader domains affects drug perturbations. (B) Immunofluorescence images of mESCs showing the nuclear localization of different valencies of the second bromodomain from BRD4 in the Parbit system (green) and their colocalization with Hoechst (magenta) after drug treatments. All scale bars are 5 µM. Drug treatments were performed at 1 μM concentrations for 24 hours. Bottom panel: Representative pseudocolored images (eGFP signal) depicting the differences in fluorescence intensities in different cell lines. A gradient pseudocolor bar (signal intensity) is shown at the left. (C) Normalized FACS data showing the effects of ARV-825 PROTAC treatment on cells expressing several combinations of bromodomains from BRD4. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

Techniques: Immunofluorescence, Fluorescence, Expressing

(A) Top: Schematic showing how the competitive binding of small molecule inhibitors versus PROTACs for the binding pocket of Acyl-eCRs can be used to measure the affinity of a small molecule for a bromodomain in cellulo . Inhibitors with higher affinity for a bromodomain, better prevent PROTAC-induced degradation. Bottom : Treatment scheme for competitive binding experiments. Cells were treated with 1 μM inhibitors for 1 hour. Then, varying concentrations of the PROTAC were added in addition to the previously added inhibitor. After 3 hours of treatment, the cell fluorescence was measured via flow cytometry. (B) Competitive binding between ARV-825 and several small molecule inhibitors showing how the inhibitors bind to BRD4(2)_BRD.1x. The cells were treated with the indicated inhibitor at a 1 μM concentration for 1 hour. Then, the stated concentration of ARV-825 PROTAC was added for 3 hours, in addition to the previous concentration of the same inhibitor. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells. (C) Competitive binding between dCBP-1 and several small molecule inhibitors showing how the inhibitors bind CBP bromodomains in Acyl-eCR constructs versus the endogenous CBP protein. The cells were treated with the indicated inhibitor at a 1 μM concentration for 1 hour. Then, the stated concentration of dCBP-1 PROTAC was added for 3 hours, in addition to the previous concentration of the same inhibitor. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

Journal: bioRxiv

Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

doi: 10.1101/2025.09.06.674632

Figure Lengend Snippet: (A) Top: Schematic showing how the competitive binding of small molecule inhibitors versus PROTACs for the binding pocket of Acyl-eCRs can be used to measure the affinity of a small molecule for a bromodomain in cellulo . Inhibitors with higher affinity for a bromodomain, better prevent PROTAC-induced degradation. Bottom : Treatment scheme for competitive binding experiments. Cells were treated with 1 μM inhibitors for 1 hour. Then, varying concentrations of the PROTAC were added in addition to the previously added inhibitor. After 3 hours of treatment, the cell fluorescence was measured via flow cytometry. (B) Competitive binding between ARV-825 and several small molecule inhibitors showing how the inhibitors bind to BRD4(2)_BRD.1x. The cells were treated with the indicated inhibitor at a 1 μM concentration for 1 hour. Then, the stated concentration of ARV-825 PROTAC was added for 3 hours, in addition to the previous concentration of the same inhibitor. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells. (C) Competitive binding between dCBP-1 and several small molecule inhibitors showing how the inhibitors bind CBP bromodomains in Acyl-eCR constructs versus the endogenous CBP protein. The cells were treated with the indicated inhibitor at a 1 μM concentration for 1 hour. Then, the stated concentration of dCBP-1 PROTAC was added for 3 hours, in addition to the previous concentration of the same inhibitor. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

Techniques: Binding Assay, Fluorescence, Flow Cytometry, Concentration Assay, Construct

(A) Phylogenetic tree showing that the panel of Acyl-eCRs comprises representative bromodomains from all classes of bromodomains. All bromodomain protein sequences were obtained from InterPro and aligned with Clustal Omega’s multiple sequence alignment. Bromodomains are classified based on structure & druggability . Black circles represent the bromodomains that are included in the panel of Acyl-eCR cell lines. (B) Normalized FACS data showing the effects of dCBP-1 PROTAC treatments on all Acyl-eCR cell lines. PROTAC was added at a 1 μM concentration for 24 hours of treatment. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

Journal: bioRxiv

Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

doi: 10.1101/2025.09.06.674632

Figure Lengend Snippet: (A) Phylogenetic tree showing that the panel of Acyl-eCRs comprises representative bromodomains from all classes of bromodomains. All bromodomain protein sequences were obtained from InterPro and aligned with Clustal Omega’s multiple sequence alignment. Bromodomains are classified based on structure & druggability . Black circles represent the bromodomains that are included in the panel of Acyl-eCR cell lines. (B) Normalized FACS data showing the effects of dCBP-1 PROTAC treatments on all Acyl-eCR cell lines. PROTAC was added at a 1 μM concentration for 24 hours of treatment. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

Techniques: Sequencing, Concentration Assay

$(A) Schematic showing how nuclei isolation & permeabilization followed by flow cytometry can measure the retention of proteins on chromatin in lieu of high background fluorescence. Nuclei can be harvested and permeabilized from whole cells, and then washed to remove the unbound or weakly bound fraction of the protein of interest. Since the weakly bound fraction of protein is removed, the fraction of protein remaining can be measured at a better signal-to-noise ratio via flow cytometry. (B) Flow cytometry analysis of nuclei harvested from BRD9_BRD.1x or WT cells. N3 gate shows the GFP signal being measured in nuclei, after iBRD9 or control treatments. Treatments in the WT cell line show a change in autofluorescence in the nuclei from the drug treatments. (C) Normalized flow cytometry data showing how Acyl-eCRs with 1x or 2x copies of the BRD9 bromodomain remain bound to chromatin, after iBRD9 treatments. iBRD9 treatments were performed at 1 μM concentration for 24 hours. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.$

Journal: bioRxiv

Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

doi: 10.1101/2025.09.06.674632

Figure Lengend Snippet: (A) Schematic showing how nuclei isolation & permeabilization followed by flow cytometry can measure the retention of proteins on chromatin in lieu of high background fluorescence. Nuclei can be harvested and permeabilized from whole cells, and then washed to remove the unbound or weakly bound fraction of the protein of interest. Since the weakly bound fraction of protein is removed, the fraction of protein remaining can be measured at a better signal-to-noise ratio via flow cytometry. (B) Flow cytometry analysis of nuclei harvested from BRD9_BRD.1x or WT cells. N3 gate shows the GFP signal being measured in nuclei, after iBRD9 or control treatments. Treatments in the WT cell line show a change in autofluorescence in the nuclei from the drug treatments. (C) Normalized flow cytometry data showing how Acyl-eCRs with 1x or 2x copies of the BRD9 bromodomain remain bound to chromatin, after iBRD9 treatments. iBRD9 treatments were performed at 1 μM concentration for 24 hours. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

Techniques: Isolation, Flow Cytometry, Fluorescence, Control, Concentration Assay

Journal: bioRxiv

Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

doi: 10.1101/2025.09.06.674632

Techniques: Isolation, Flow Cytometry, Fluorescence, Control, Concentration Assay

Inhibition of BRD9 enhances the induction of PPARα-downstream gene CPT1A by WY-14643. A: Schematic image of mammalian cBAF, PBAF, and ncBAF . B: Glycerol gradient density sedimentation of the nuclear extract of HepG2 cells. Subunits of each SWI/SNF complex and PPARα protein were detected by Western blot analysis (C–G) HepG2, PHH, PMH, or HepaSH cells were pre-treated with 20 μM BI-9564. After 12 h, the cells were co-treated with 10 μM WY-14643 and 20 μM BI-9564 for 48 h. Human (C and E) and mouse (F) CPT1A mRNA levels were determined using real-time RT-PCR. (D) CPT1A, PPARα, and GAPDH protein levels in HepG2 cells were determined by Western blotting. Each column represents the mean ± SD (n = 4). Western blot experiments were conducted with three independent replicates. In panel (C), data were analyzed using one-way ANOVA followed by the Games-Howell test. In panel (F), data were analyzed using the Kruskal–Wallis test followed by Dunn’s test. Data in all other panels were analyzed by one-way ANOVA followed by Tukey’s test. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001, compared with NT. † P < 0.05, †† P < 0.01 and ††† P < 0.001, compared with BI-9564 (−). NT: non-treatment.

Journal: Journal of Lipid Research

Article Title: Chromatin remodeler BRD9 represses transcription of PPARα target genes, including CPT1A to suppress lipid metabolism

doi: 10.1016/j.jlr.2025.100874

Figure Lengend Snippet: Inhibition of BRD9 enhances the induction of PPARα-downstream gene CPT1A by WY-14643. A: Schematic image of mammalian cBAF, PBAF, and ncBAF . B: Glycerol gradient density sedimentation of the nuclear extract of HepG2 cells. Subunits of each SWI/SNF complex and PPARα protein were detected by Western blot analysis (C–G) HepG2, PHH, PMH, or HepaSH cells were pre-treated with 20 μM BI-9564. After 12 h, the cells were co-treated with 10 μM WY-14643 and 20 μM BI-9564 for 48 h. Human (C and E) and mouse (F) CPT1A mRNA levels were determined using real-time RT-PCR. (D) CPT1A, PPARα, and GAPDH protein levels in HepG2 cells were determined by Western blotting. Each column represents the mean ± SD (n = 4). Western blot experiments were conducted with three independent replicates. In panel (C), data were analyzed using one-way ANOVA followed by the Games-Howell test. In panel (F), data were analyzed using the Kruskal–Wallis test followed by Dunn’s test. Data in all other panels were analyzed by one-way ANOVA followed by Tukey’s test. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001, compared with NT. † P < 0.05, †† P < 0.01 and ††† P < 0.001, compared with BI-9564 (−). NT: non-treatment.

Article Snippet: Rabbit polyclonal antibody for human BRD9 (A303-781A) was purchased from Bethyl Laboratories.

Techniques: Inhibition, Sedimentation, Western Blot, Quantitative RT-PCR

Effects of BRD7 and BRD9 knockdown on CPT1A expression in WY-14643-treated HepG2 cells. A–C: HepG2 cells were transfected with siRNA against BRD7 (siBRD7) or BRD9 (siBRD9). After 24 h, the cells were treated with 30 μM WY-14643 for 48 h. A: BRD7, BRD9, PPARα, and GAPDH protein levels were determined using Western blot analysis. B and C: CPT1A mRNA and protein levels were determined using real-time RT-PCR and Western blotting analysis, respectively. Each column represents the mean ± SD (n = 4). Western blot experiments were conducted with three independent replicates. Data in all panels were analyzed by one-way ANOVA followed by Tukey’s test. ∗∗ P < 0.01 and ∗∗∗ P < 0.001, compared with NT, † P < 0.05 and ††† P < 0.001, compared with siControl. NT: non-treatment.

Journal: Journal of Lipid Research

Article Title: Chromatin remodeler BRD9 represses transcription of PPARα target genes, including CPT1A to suppress lipid metabolism

doi: 10.1016/j.jlr.2025.100874

Figure Lengend Snippet: Effects of BRD7 and BRD9 knockdown on CPT1A expression in WY-14643-treated HepG2 cells. A–C: HepG2 cells were transfected with siRNA against BRD7 (siBRD7) or BRD9 (siBRD9). After 24 h, the cells were treated with 30 μM WY-14643 for 48 h. A: BRD7, BRD9, PPARα, and GAPDH protein levels were determined using Western blot analysis. B and C: CPT1A mRNA and protein levels were determined using real-time RT-PCR and Western blotting analysis, respectively. Each column represents the mean ± SD (n = 4). Western blot experiments were conducted with three independent replicates. Data in all panels were analyzed by one-way ANOVA followed by Tukey’s test. ∗∗ P < 0.01 and ∗∗∗ P < 0.001, compared with NT, † P < 0.05 and ††† P < 0.001, compared with siControl. NT: non-treatment.

Article Snippet: Rabbit polyclonal antibody for human BRD9 (A303-781A) was purchased from Bethyl Laboratories.

Techniques: Knockdown, Expressing, Transfection, Western Blot, Quantitative RT-PCR

BRD9 directly interacts with PPARα. A: Co-immunoprecipitation assay to examine the interaction between PPARα and BRD9 was performed. HEK293T cells were transfected with FLAG-PPARα plasmid together with the BRD7-His or BRD9-His plasmids using Lipofectamine 3000. Cell lysates were subjected to co-immunoprecipitation with His-tag antibody. B: Direct interaction between PPARα and BRD9. HEK293T cells were individually transfected with FLAG-PPARα, BRD7-His, or BRD9-His plasmids. Affinity purification was conducted using cell lysates, and the purified FLAG-PPAR protein was incubated with purified BRD7/9-His proteins. C: Western blotting to analyze lysine acetylation of PPARα. Lysate of FLAG-PPARα plasmid-transfected HEK293T cells was subjected to Western blotting using anti-acetylated lysine and anti-FLAG antibodies. A smaller amount of protein (10 μg) was loaded compared to A (30 μg). Western blot experiments were conducted with three independent replicates.

Journal: Journal of Lipid Research

Article Title: Chromatin remodeler BRD9 represses transcription of PPARα target genes, including CPT1A to suppress lipid metabolism

doi: 10.1016/j.jlr.2025.100874

Figure Lengend Snippet: BRD9 directly interacts with PPARα. A: Co-immunoprecipitation assay to examine the interaction between PPARα and BRD9 was performed. HEK293T cells were transfected with FLAG-PPARα plasmid together with the BRD7-His or BRD9-His plasmids using Lipofectamine 3000. Cell lysates were subjected to co-immunoprecipitation with His-tag antibody. B: Direct interaction between PPARα and BRD9. HEK293T cells were individually transfected with FLAG-PPARα, BRD7-His, or BRD9-His plasmids. Affinity purification was conducted using cell lysates, and the purified FLAG-PPAR protein was incubated with purified BRD7/9-His proteins. C: Western blotting to analyze lysine acetylation of PPARα. Lysate of FLAG-PPARα plasmid-transfected HEK293T cells was subjected to Western blotting using anti-acetylated lysine and anti-FLAG antibodies. A smaller amount of protein (10 μg) was loaded compared to A (30 μg). Western blot experiments were conducted with three independent replicates.

Article Snippet: Rabbit polyclonal antibody for human BRD9 (A303-781A) was purchased from Bethyl Laboratories.

Techniques: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Affinity Purification, Purification, Incubation, Western Blot

Effects of WY-14643 and/or BI-9564 treatment on the binding of PPARα and BRD9 and on the chromatin accessibility around the CPT1A intronic peroxisomal proliferator response element (PPRE). A: Scheme of the CPT1A gene. B: Binding of PPARα to CPT1A intronic PPRE. HepG2 cells were pre-treated with 20 μM BI-9564. After 12 h, the cells were co-treated with 10 μM WY-14643 and 20 μM BI-9564 for 48 h. A ChIP assay using an anti-PPARα antibody was performed, and the purified DNA was analyzed by real-time PCR targeting CPT1A intronic PPRE. C: The interaction between PPARα and BRD9. HEK293T cells were transfected with FLAG-PPARα and BRD9-His plasmids using Lipofectamine 3000, and treated with 30 μM WY-14643 and/or 40 μM BI-9564 for 48 h. The cell lysates were co-immunoprecipitated with anti-His-tag antibody. FLAG-PPARα and BRD9-His were detected by Western blotting. D: Chromatin accessibility around CPT1A intronic PPRE. HepG2 cells were pre-treated with 20 μM BI-9564. After 12 h, the cells were co-treated with 10 μM WY-14643 and 20 μM BI-9564 for 48 h; a FAIRE assay was then performed on the cells. Purified DNA was analyzed by real-time PCR targeting the CPT1A intronic PPRE. Each column represents the mean ± SD (n = 3). Data in all panels were analyzed by one-way ANOVA followed by Tukey’s test. ∗∗∗ P < 0.001, compared with NT; † P < 0.05 and ††† P < 0.001, compared with BI-9564 (−). NT: non-treatment.

Journal: Journal of Lipid Research

Article Title: Chromatin remodeler BRD9 represses transcription of PPARα target genes, including CPT1A to suppress lipid metabolism

doi: 10.1016/j.jlr.2025.100874

Figure Lengend Snippet: Effects of WY-14643 and/or BI-9564 treatment on the binding of PPARα and BRD9 and on the chromatin accessibility around the CPT1A intronic peroxisomal proliferator response element (PPRE). A: Scheme of the CPT1A gene. B: Binding of PPARα to CPT1A intronic PPRE. HepG2 cells were pre-treated with 20 μM BI-9564. After 12 h, the cells were co-treated with 10 μM WY-14643 and 20 μM BI-9564 for 48 h. A ChIP assay using an anti-PPARα antibody was performed, and the purified DNA was analyzed by real-time PCR targeting CPT1A intronic PPRE. C: The interaction between PPARα and BRD9. HEK293T cells were transfected with FLAG-PPARα and BRD9-His plasmids using Lipofectamine 3000, and treated with 30 μM WY-14643 and/or 40 μM BI-9564 for 48 h. The cell lysates were co-immunoprecipitated with anti-His-tag antibody. FLAG-PPARα and BRD9-His were detected by Western blotting. D: Chromatin accessibility around CPT1A intronic PPRE. HepG2 cells were pre-treated with 20 μM BI-9564. After 12 h, the cells were co-treated with 10 μM WY-14643 and 20 μM BI-9564 for 48 h; a FAIRE assay was then performed on the cells. Purified DNA was analyzed by real-time PCR targeting the CPT1A intronic PPRE. Each column represents the mean ± SD (n = 3). Data in all panels were analyzed by one-way ANOVA followed by Tukey’s test. ∗∗∗ P < 0.001, compared with NT; † P < 0.05 and ††† P < 0.001, compared with BI-9564 (−). NT: non-treatment.

Article Snippet: Rabbit polyclonal antibody for human BRD9 (A303-781A) was purchased from Bethyl Laboratories.

Techniques: Binding Assay, Purification, Real-time Polymerase Chain Reaction, Transfection, Immunoprecipitation, Western Blot

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