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

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

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

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

Journal: The Journal of Biological Chemistry

Article Title: Glioma tumor suppressor candidate region gene 1 (GLTSCR1) and its paralog GLTSCR1-like form SWI/SNF chromatin remodeling subcomplexes

doi: 10.1074/jbc.RA117.001065

Figure Lengend Snippet: GLTSCR1 is in a novel SWI/SNF subcomplex GBAF. A, Glycerol gradients from renal cancer cell line Caki1 and prostate cancer cell line PC3 indicate that GLTSCR1 does not co-sediment with BAF subunit ARID1A or PBAF subunits PBRM1 (for Caki1) or BRD7 (for PC3). B, IP experiments of BAF subunits from PC3 lysates identify GLTSCR1 association with BRG1 and BRM. C, BAF subunit and GLTSCR1 IP experiments from HEK293T lysates identify GLTSCR1 association with BAF155 and BAF53a but not BAF47, BAF57, or BAF45D. D, BAF subunit and GLTSCR1 IP experiments from HEK293T lysates identify GLTSCR1 association with SS18 and BRD9 but not BCL11A. E and F, glycerol gradient analysis (E) and BAF subunit IP experiments (F) from HEK293T lysates identify GLTSCR1 association with BRG1 and SS18 but not BAF170 and BAF47 and validate BRD9 as a subunit found in GBAF, but not BAF/PBAF. G, schematic representation of GBAF, BAF, and PBAF composition. Yellow subunits are unique to GBAF, blue subunits are unique to BAF, red subunits are unique to PBAF, green subunits are shared by GBAF and BAF, purple subunits are shared by BAF and PBAF, and gray subunits are shared by all three complexes. Subcomplex GBAF consists of BAF60A, BRG1, BAF155, BRD9, BAF53A, and SS18. H, GBAF possesses ATPase activity. ATPase activity assay was performed with BRG1 and GLTSCR1 immunoprecipitations providing similar levels of BRG1. ATPase activities normalized to respective IgG isotype controls yielded comparable fold changes (3.03 ± 0.23, for BRG1 IP; 3.24 ± 0.87, for GLTSCR1 IP). Error bars, means ± S.D. (n = 3). *, p < 0.05; ***, p < 0.001. I, sequential salt extraction analysis and immunoblot quantitation indicates that GLTSCR1 interacts with bulk chromatin at a similar strength as ARID1A (representative of BAF) and PBRM1 (representative of PBAF).

Article Snippet: Antibodies Antibodies used in the study are BRG1 (Abcam, ab110641, IP, and Western blotting), BAF60A (Bethyl, A301-594A, IP), BAF170 (Santa Cruz, sc-17838, IP, and Western blotting), GLTSCR1 (Santa Cruz, sc-515086, IP, and Western blotting), FLAG (Sigma–Aldrich, F1804), BRD4 (Bethyl, A301-985A50, IP), BRD9 (A303-781A IP and Western blotting), SS18 (Cell Signaling Technologies, 21792S, IP, and Western blotting), ARID1A (Santa Cruz, sc-32761, Western blotting), GLTSCR1L/BICRAL (Invitrogen, PA5-56126, Western blotting), BRD4 (Bethyl, A700-005-T, Western blotting), BRD7 (Santa Cruz, sc-376180, Western blotting), ARID2 (Bethyl, A302-230A, Western blotting), BAF155 (in-house, IP) BAF45D (in-house, IP, and Western blotting), BAF57 (Bethyl, A300-810A, IP, and Western blotting), BAF155 (Santa Cruz, sc-32763, Western blotting), BAF53A (Abcam, ab131272, Western blotting), BAF60A (Santa Cruz, sc-514400, Western blotting), PBRM1 (Bethyl, A301-590A, Western blotting), ARID1B (Bethyl, A301-047-T, Western blotting), BCL11A (Santa Cruz, sc-514842, IP and Western blotting), Actin (Santa Cruz, sc-47778, Western blotting), GAPDH (Santa Cruz, sc-137179, Western blotting), and α-tubulin (Santa Cruz, sc-8035, Western blotting).

Techniques: Activity Assay, Extraction, Western Blot, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Glioma tumor suppressor candidate region gene 1 (GLTSCR1) and its paralog GLTSCR1-like form SWI/SNF chromatin remodeling subcomplexes

doi: 10.1074/jbc.RA117.001065

Figure Lengend Snippet: GBAF contains GLTSCR1 or paralog GLTSCR1L (BICRAL). A, glycerol gradient analysis in mESCs showing that GBAF-associated BRG1 and BAF60A were preserved in fractions 11–13 in the absence of GLTSCR1, suggesting that GBAF was not completely disrupted by Gltscr1 knockout. B, pairwise alignment of amino acid sequences of GLTSCR1 and its paralog GLTSCR1L (BICRAL) show homology in the N-terminal region and strong homology at region identified at a conserved GLTSCR1 domain. C, verification of inducible expression of BICRAL-FLAG in HEK293T cells with both FLAG and endogenous BICRAL antibodies. D, immunoprecipitation analysis showed that similar to GLTSCR1, exogenous BICRAL interacts with BRD9, BAF53A, and BRG1. In addition, endogenous GLTSCR1 and BICRAL do not immunoprecipitate one another. BICRAL overexpression results in reduced GLTSCR1 protein levels. E, endogenous BICRAL does not associate with GLTSCR1, further validating that GLTSCR1 and BICRAL are mutually exclusive in GBAF context. BICRAL is detected in total BRG1 IP but not in GLTSCR1 IP, although both contain comparable levels of BRG1. Note that the same Western blotting is used in Fig. S1B to compare BRG1 levels for ATPase assay.

Article Snippet: Antibodies Antibodies used in the study are BRG1 (Abcam, ab110641, IP, and Western blotting), BAF60A (Bethyl, A301-594A, IP), BAF170 (Santa Cruz, sc-17838, IP, and Western blotting), GLTSCR1 (Santa Cruz, sc-515086, IP, and Western blotting), FLAG (Sigma–Aldrich, F1804), BRD4 (Bethyl, A301-985A50, IP), BRD9 (A303-781A IP and Western blotting), SS18 (Cell Signaling Technologies, 21792S, IP, and Western blotting), ARID1A (Santa Cruz, sc-32761, Western blotting), GLTSCR1L/BICRAL (Invitrogen, PA5-56126, Western blotting), BRD4 (Bethyl, A700-005-T, Western blotting), BRD7 (Santa Cruz, sc-376180, Western blotting), ARID2 (Bethyl, A302-230A, Western blotting), BAF155 (in-house, IP) BAF45D (in-house, IP, and Western blotting), BAF57 (Bethyl, A300-810A, IP, and Western blotting), BAF155 (Santa Cruz, sc-32763, Western blotting), BAF53A (Abcam, ab131272, Western blotting), BAF60A (Santa Cruz, sc-514400, Western blotting), PBRM1 (Bethyl, A301-590A, Western blotting), ARID1B (Bethyl, A301-047-T, Western blotting), BCL11A (Santa Cruz, sc-514842, IP and Western blotting), Actin (Santa Cruz, sc-47778, Western blotting), GAPDH (Santa Cruz, sc-137179, Western blotting), and α-tubulin (Santa Cruz, sc-8035, Western blotting).

Techniques: Knock-Out, Expressing, Immunoprecipitation, Over Expression, Western Blot, ATPase Assay

Journal: The Journal of Biological Chemistry

Article Title: Glioma tumor suppressor candidate region gene 1 (GLTSCR1) and its paralog GLTSCR1-like form SWI/SNF chromatin remodeling subcomplexes

doi: 10.1074/jbc.RA117.001065

Figure Lengend Snippet: GLTSCR1 and BICRAL are mutually exclusive subunits of GBAF that can alter SWI/SNF complex stoichiometry. A, glycerol gradient analysis in BICRAL-FLAG–overexpressing HEK293T cells indicates that BICRAL is incorporated into GBAF. Overexpression of BICRAL-FLAG increases the GBAF-associated BRG1 levels (fractions 11–14), suggesting formation of new GBAF upon BICRAL overexpression. Reduced GBAF-associated GLTSCR1 levels also validate decreased GLTSCR1 protein expression upon BICRAL overexpression. B, immunoprecipitation analysis showing that BICRAL-FLAG overexpression reduced BRG1-associated GLTSCR1 levels and enhanced BRD9 protein levels and its association with BRG1. BICRAL-FLAG overexpression also reduced BRG1-associated BAF47 and BAF57, suggesting competition between GBAF and BAF for BRG1. C, RT-qPCR showing that expression of BRG1, GLTSCR1, or BRD9 did not alter upon BICRAL overexpression. Error bars, means ± S.D. (n = 3). D, CRISPR/Cas9-mediated knockout of GLTSCR1 with or without CRISPR/Cas9-mediated knockout of BICRAL reduced the BRG1-associated BRD9 levels, as an indicator of loss of GBAF.

Article Snippet: Antibodies Antibodies used in the study are BRG1 (Abcam, ab110641, IP, and Western blotting), BAF60A (Bethyl, A301-594A, IP), BAF170 (Santa Cruz, sc-17838, IP, and Western blotting), GLTSCR1 (Santa Cruz, sc-515086, IP, and Western blotting), FLAG (Sigma–Aldrich, F1804), BRD4 (Bethyl, A301-985A50, IP), BRD9 (A303-781A IP and Western blotting), SS18 (Cell Signaling Technologies, 21792S, IP, and Western blotting), ARID1A (Santa Cruz, sc-32761, Western blotting), GLTSCR1L/BICRAL (Invitrogen, PA5-56126, Western blotting), BRD4 (Bethyl, A700-005-T, Western blotting), BRD7 (Santa Cruz, sc-376180, Western blotting), ARID2 (Bethyl, A302-230A, Western blotting), BAF155 (in-house, IP) BAF45D (in-house, IP, and Western blotting), BAF57 (Bethyl, A300-810A, IP, and Western blotting), BAF155 (Santa Cruz, sc-32763, Western blotting), BAF53A (Abcam, ab131272, Western blotting), BAF60A (Santa Cruz, sc-514400, Western blotting), PBRM1 (Bethyl, A301-590A, Western blotting), ARID1B (Bethyl, A301-047-T, Western blotting), BCL11A (Santa Cruz, sc-514842, IP and Western blotting), Actin (Santa Cruz, sc-47778, Western blotting), GAPDH (Santa Cruz, sc-137179, Western blotting), and α-tubulin (Santa Cruz, sc-8035, Western blotting).

Techniques: Over Expression, Expressing, Immunoprecipitation, Quantitative RT-PCR, CRISPR, Knock-Out

Journal: The Journal of Biological Chemistry

Article Title: Glioma tumor suppressor candidate region gene 1 (GLTSCR1) and its paralog GLTSCR1-like form SWI/SNF chromatin remodeling subcomplexes

doi: 10.1074/jbc.RA117.001065

Figure Lengend Snippet: GLTSCR1 associates with BRD4 but is not required for BRD4-mediated MYC transcription in LNCaP cells. A, immunoprecipitation of BRD4 enriches GLTSCR1, BRD9, and BAF155 but not BAF/PBAF subunit BAF47. AP, lysates treated with alkaline phosphatase; PI, lysates treated with phosphatase inhibitors. B, proliferation measurement after 6 days of growth of LNCaP cells with GLTSCR1 knockout using Alamar Blue. Error bars, means ± S.D. for n = 6 replicates. C, GLTSCR1 knockout sensitized LNCaP to BET inhibitor JQ1. Cell numbers are approximated using Alamar Blue fluorescence. IC50 values are derived from curve fit calculations using GraphPad Prism and presented as means ± S.D. for n = 4 replicates. **, p < 0.01. D, MYC expression is up-regulated in GLTSCR1 knockout LNCaP cells, which reverted back to basal levels upon 50 nm JQ1 treatment. Error bars, mean ± S.D. (n = 3 replicates). *, p < 0.05.

Article Snippet: Antibodies Antibodies used in the study are BRG1 (Abcam, ab110641, IP, and Western blotting), BAF60A (Bethyl, A301-594A, IP), BAF170 (Santa Cruz, sc-17838, IP, and Western blotting), GLTSCR1 (Santa Cruz, sc-515086, IP, and Western blotting), FLAG (Sigma–Aldrich, F1804), BRD4 (Bethyl, A301-985A50, IP), BRD9 (A303-781A IP and Western blotting), SS18 (Cell Signaling Technologies, 21792S, IP, and Western blotting), ARID1A (Santa Cruz, sc-32761, Western blotting), GLTSCR1L/BICRAL (Invitrogen, PA5-56126, Western blotting), BRD4 (Bethyl, A700-005-T, Western blotting), BRD7 (Santa Cruz, sc-376180, Western blotting), ARID2 (Bethyl, A302-230A, Western blotting), BAF155 (in-house, IP) BAF45D (in-house, IP, and Western blotting), BAF57 (Bethyl, A300-810A, IP, and Western blotting), BAF155 (Santa Cruz, sc-32763, Western blotting), BAF53A (Abcam, ab131272, Western blotting), BAF60A (Santa Cruz, sc-514400, Western blotting), PBRM1 (Bethyl, A301-590A, Western blotting), ARID1B (Bethyl, A301-047-T, Western blotting), BCL11A (Santa Cruz, sc-514842, IP and Western blotting), Actin (Santa Cruz, sc-47778, Western blotting), GAPDH (Santa Cruz, sc-137179, Western blotting), and α-tubulin (Santa Cruz, sc-8035, Western blotting).

Techniques: Immunoprecipitation, Knock-Out, Fluorescence, Derivative Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Glioma tumor suppressor candidate region gene 1 (GLTSCR1) and its paralog GLTSCR1-like form SWI/SNF chromatin remodeling subcomplexes

doi: 10.1074/jbc.RA117.001065

Figure Lengend Snippet: GLTSCR1 and BICRAL are expressed in most cell lines but are uniquely required for the viability of prostate cancer cell line PC3. A, immunoblot analysis of GLTSCR1 and BICRAL expression across a panel of cell lines. B, proliferation measurement after 6 days of growth of the non-transformed mouse cell lines mESCs and NMuMG with Gltscr1 knockout using Alamar Blue. C, proliferation measurement after 6 days of growth of the transformed human astrocyte cell line SVGp12 and glioblastoma cell line T98G with GLTSCR1 knockout using Alamar Blue. D, proliferation measurement after 6 days of growth of HEK293T cells with GLTSCR1 and BICRAL knockout using Alamar Blue. E, validation of knockouts using multiple guide RNAs. F, left panel, Alamar Blue assay demonstrated that loss of GLTSCR1 and BICRAL reduced the growth of PC3 cells 6 day after plating. Fluorescence values graphed (excitation, 560 nm; emission, 590 nm) represent the metric for cell number. Error bars, means ± S.D. (n = 3 biological replicates). **, p < 0.01; ***, p < 0.001 compared with control cells. Right panel, loss of GLTSCR1 reduced the clonogenic growth of prostate cell line PC3. G, PC3 cells did not display sensitivity to BRD9 inhibitor BI-7273 (IC50 of 275 nm) up to 10 μm treatment for 4 days. Cell number was approximated using Alamar Blue fluorescence (n = 3 biological replicates).

Article Snippet: Antibodies Antibodies used in the study are BRG1 (Abcam, ab110641, IP, and Western blotting), BAF60A (Bethyl, A301-594A, IP), BAF170 (Santa Cruz, sc-17838, IP, and Western blotting), GLTSCR1 (Santa Cruz, sc-515086, IP, and Western blotting), FLAG (Sigma–Aldrich, F1804), BRD4 (Bethyl, A301-985A50, IP), BRD9 (A303-781A IP and Western blotting), SS18 (Cell Signaling Technologies, 21792S, IP, and Western blotting), ARID1A (Santa Cruz, sc-32761, Western blotting), GLTSCR1L/BICRAL (Invitrogen, PA5-56126, Western blotting), BRD4 (Bethyl, A700-005-T, Western blotting), BRD7 (Santa Cruz, sc-376180, Western blotting), ARID2 (Bethyl, A302-230A, Western blotting), BAF155 (in-house, IP) BAF45D (in-house, IP, and Western blotting), BAF57 (Bethyl, A300-810A, IP, and Western blotting), BAF155 (Santa Cruz, sc-32763, Western blotting), BAF53A (Abcam, ab131272, Western blotting), BAF60A (Santa Cruz, sc-514400, Western blotting), PBRM1 (Bethyl, A301-590A, Western blotting), ARID1B (Bethyl, A301-047-T, Western blotting), BCL11A (Santa Cruz, sc-514842, IP and Western blotting), Actin (Santa Cruz, sc-47778, Western blotting), GAPDH (Santa Cruz, sc-137179, Western blotting), and α-tubulin (Santa Cruz, sc-8035, Western blotting).

Techniques: Western Blot, Expressing, Transformation Assay, Knock-Out, Biomarker Discovery, Alamar Blue Assay, Fluorescence, Control

Journal: bioRxiv

Article Title: ncBAF, a chromatin remodeler, enhances PXR-mediated transcriptional activation in the human and mouse liver

doi: 10.1101/2023.02.03.527063

Figure Lengend Snippet: Identification of proteins interacting with PXR. ShP51 cells were treated with rifampicin for 24 hr. Co-immunoprecipitation with anti-PXR antibody was performed using whole cell lysates followed by mass spectrometric analysis. (A) The numbers of proteins detected in the immunoprecipitant of rifampicin-and/or non-treated cells are shown in a Venn diagram. The ratios of the protein amounts of the rifampicin-treated sample to those of non-treated sample are shown in the bottom graph. (B) The proteins whose abundance ratio rifampicin-treated/non-treated was > 2.0 (168 proteins) as well as proteins detected only in rifampicin-treated sample (804 proteins) were subjected to pathway analysis using DAVID Bioinformatics Resources ( https://david.ncifcrf.gov/home.jsp ). (C) Schematic representation of BAF, PBAF, and ncBAF composition reported by ref. 21 and ref. 58. (D) Glycerol gradient sedimentation was performed using ShP51 cell nuclear extracts, and ARID1A, BRD7, BRD9, and PXR proteins were detected by Western blotting. Source data are available online for this figure.

Article Snippet: Rabbit anti-human BRD9 polyclonal antibody (A303-781A), rabbit anti-ARID1A (GTX129433) antibody, and rabbit anti-SMARCD1 (BAF60A) antibody (10998-2-AP) were obtained from Bethyl Laboratories (Montgomery, AL), Genetex (Irvine, CA), and Proteintech (Rosemont, IL), respectively.

Techniques: Immunoprecipitation, Sedimentation, Western Blot

Journal: bioRxiv

Article Title: ncBAF, a chromatin remodeler, enhances PXR-mediated transcriptional activation in the human and mouse liver

doi: 10.1101/2023.02.03.527063

Figure Lengend Snippet: Interaction between PXR and BRD9. HEK293T cells were transfected with FLAG-PXR along with BRD7-His or BRD9-His plasmid (A), transfected with BRD9-His along with FLAG-PXR or FLAG-PXR K109A plasmid (B), or transfected with FLAG-PXR and BRD9-His plasmids followed by treatment with rifampicin along with iBRD9 for 1 hr (C). ShP51 cells were treated with rifampicin along with iBRD9 for 24 hr (D). (A, B, and C) Immunoprecipitation with anti-FLAG antibody was performed using whole HEK293T cell lysates, and FLAG and His tags were detected by Western blotting. (D) Immunoprecipitation with anti-PXR antibody was performed using ShP51 cell nuclear extracts, and BRD9 and PXR proteins were detected by Western blotting. IP: immunoprecipitation, WB: Western blotting. The experiments were repeated two times with similar results.

Article Snippet: Rabbit anti-human BRD9 polyclonal antibody (A303-781A), rabbit anti-ARID1A (GTX129433) antibody, and rabbit anti-SMARCD1 (BAF60A) antibody (10998-2-AP) were obtained from Bethyl Laboratories (Montgomery, AL), Genetex (Irvine, CA), and Proteintech (Rosemont, IL), respectively.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: ncBAF, a chromatin remodeler, enhances PXR-mediated transcriptional activation in the human and mouse liver

doi: 10.1101/2023.02.03.527063

Figure Lengend Snippet: Effects of BRD7 or BRD9 knockdown or BRD9 inhibition on PXR-mediated induction of CYP3A4 expression. (A) ShP51 cells were transfected with siRNA for BRD7 (siBRD7) or BRD9 (siBRD9). After incubation for 24 hr, the cells were treated with 10 μM rifampicin or 10 μM simvastatin. ShP51 cells (B), HepaRG cells (C), and human primary hepatocytes (D and E) were treated with rifampicin or simvastatin along with iBRD9. PXR, RXRα and GAPDH (A and B) protein, CYP3A4 and GAPDH mRNA (A-D), and CYP3A4 enzyme activity (E) were evaluated by Western blotting, real-time RT□PCR, and P450-Glo assay. Each column represents the mean ± SD (n =3-4). n refers to biological repeats. * 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 siControl or iBRD9 (−). NT: non-treatment. The experiments were repeated two times with similar results.

Article Snippet: Rabbit anti-human BRD9 polyclonal antibody (A303-781A), rabbit anti-ARID1A (GTX129433) antibody, and rabbit anti-SMARCD1 (BAF60A) antibody (10998-2-AP) were obtained from Bethyl Laboratories (Montgomery, AL), Genetex (Irvine, CA), and Proteintech (Rosemont, IL), respectively.

Techniques: Knockdown, Inhibition, Expressing, Transfection, Incubation, Activity Assay, Western Blot, Glo Assay

Journal: bioRxiv

Article Title: ncBAF, a chromatin remodeler, enhances PXR-mediated transcriptional activation in the human and mouse liver

doi: 10.1101/2023.02.03.527063

Figure Lengend Snippet: Effects of iBRD9 on PXR binding to chromatin and changes in chromatin structure. (A) ShP51 cells were treated with 20 μM rifampicin along with 20 μM iBRD9 for 24 hr. SSE analysis followed by Western blotting for BRD9 or PXR was performed. The peaks of band intensity are shown in red. (B) Schematic representation of the upstream of CYP3A4 gene. ShP51 cells were treated with 10 μM rifampicin along with 10 μM iBRD9 for 24 hr. Immunoprecipitation with anti-PXR antibody (C) or anti-BRD9 antibody (D) was performed using chromatin from ShP51 cells. Enrichment of the proximal promoter, distal enhancer, and far enhancer of CYP3A4 was evaluated by real-time PCR. (E) Changes in chromatin structure was evaluated by FAIRE followed by real-time PCR. Each column represents the mean ± SD (n = 3). n refers to biological repeats. * P < 0.05, ** P < 0.01, and *** P < 0.001, compared with NT, †† P < 0.01 and ††† P < 0.001, compared with iBRD9 (−). NT: non-treatment. The experiments were repeated two times with similar results.

Article Snippet: Rabbit anti-human BRD9 polyclonal antibody (A303-781A), rabbit anti-ARID1A (GTX129433) antibody, and rabbit anti-SMARCD1 (BAF60A) antibody (10998-2-AP) were obtained from Bethyl Laboratories (Montgomery, AL), Genetex (Irvine, CA), and Proteintech (Rosemont, IL), respectively.

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