Journal: Cell Genomics

Article Title: Pooled endogenous protein tagging and recruitment for systematic profiling of protein function

doi: 10.1016/j.xgen.2024.100651

Figure Lengend Snippet: Comparison of two orthogonal effector screens highlights the CTLH complex for its potential to degrade recruited substrates (A) Strategy for performing two orthogonal screens for functional effectors of degradation by targeting either mClover3-FKBP (top path) or endogenous BRD4 (bottom path). Degraders of mClover3-FKBP are enriched by FACS, while degraders of BRD4 are depleted during cell growth. (B) Cell viability assay (MTS) in HAP1 cells treated with a BRD4 inhibitor (JQ1, max growth inhibition 88%, 50% effective dose at 144 nM) or degrader (dBET6, max growth inhibition 99%, 50% effective dose at 136 nM) for 72 h. Error bars represent standard deviation. (C) Chemical structure of HaloBR-6. (D and E) Abundance versus fold change plots for tagging sgRNAs in the (D) fluorescence- or (E) growth-based screen. In (D), the ratio of sgRNAs in bright versus dim mClover3 populations is compared between cells treated with HaloFK-7 or HaloFK-2. All sgRNAs representing non-targeting controls (NTCs) are colored in blue, and screen hits (false discovery rate [FDR] < 0.05 for fluorescence- and FDR < 0.2 for growth-based screen) are orange. The CTLH complex and some effectors previously utilized for TPD are highlighted. (F) STRING association map of 57 proteins identified as top hits in at least two of the three growth screen time points. Edge thickness correlates with likelihood of the interaction.

Article Snippet: The following antibodies were used for immunoblotting: BRD4 (NBP2-76393, Novus Biologicals; 1:1000), GFP (sc-9996, Santa Cruz Biotechnology; 1:1666), alpha-tubulin (NB100-690, Novus Biologicals; 1:5000), IRDye 680LT Goat anti-Rabbit (LI-COR 926–68021, 1:10,000), IRDye 800CW Goat anti-Mouse (LI-COR 926–32210, 1:10,000).

Techniques: Comparison, Functional Assay, Viability Assay, Inhibition, Standard Deviation, Fluorescence

Journal: Cell Genomics

Article Title: Pooled endogenous protein tagging and recruitment for systematic profiling of protein function

doi: 10.1016/j.xgen.2024.100651

Figure Lengend Snippet: Members of the CTLH complex can function as flexible effectors for TPD (A) AlphaFold2-predicted structure, using ColabFold, for WDR26 fused with HaloTag at intron 6. (B) Flow cytometry plots of monoclonal WDR26-or RANBP9-tagged HAP1 cells treated with the indicated drugs for 72 h (fluorescence levels are shown in log scale). (C and D) mClover3-FKBP mean fluorescence intensity (MFI) after treatment with HaloFK-7 for (C) 24 h at indicated concentrations or at (D) 20 nM HaloFK-7 for the indicated amounts of time. The key is applicable to both subfigures. Error bars represent standard deviation. (E) Immunoblot representing 24 h degradation of BRD4 or mClover3-FKBP with indicated concentrations of HaloBR-6 or 20 nM HaloFK-7, respectively, in cells with tagged WDR26 or an inactive tag or in untagged cells (parental). (F) Counts of cells with tagged WDR26 or an inactive tag after treatment with 100 nM HaloBR-6 for the indicated amounts of time. Biological triplicates are shown and represented by box and whisker plots. (G) Ratio of mClover3-FKBP MFI between tagged cells treated with 20 nM HaloFK-7 or DMSO for 16 h in the presence of 5 μM MG132 or vehicle control. Asterisks represent p values. Error bars represent standard deviation. (H) Ratio of mClover3-FKBP MFI between tagged cells treated with 20 nM HaloFK-7 or DMSO in the presence of the indicated knockout. 24 and 72 h treatments were used for WDR26- and RANBP9-tagged cells, respectively. Red points represent unique sgRNAs for knockout. Adjusted p values are indicated above bars. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Error bars represent standard deviation. (I) Illustration of CTLH complex.

Article Snippet: The following antibodies were used for immunoblotting: BRD4 (NBP2-76393, Novus Biologicals; 1:1000), GFP (sc-9996, Santa Cruz Biotechnology; 1:1666), alpha-tubulin (NB100-690, Novus Biologicals; 1:5000), IRDye 680LT Goat anti-Rabbit (LI-COR 926–68021, 1:10,000), IRDye 800CW Goat anti-Mouse (LI-COR 926–32210, 1:10,000).

Techniques: Flow Cytometry, Fluorescence, Standard Deviation, Western Blot, Whisker Assay, Control, Knock-Out

Journal: Cell Genomics

Article Title: Pooled endogenous protein tagging and recruitment for systematic profiling of protein function

doi: 10.1016/j.xgen.2024.100651

Figure Lengend Snippet:

Article Snippet: The following antibodies were used for immunoblotting: BRD4 (NBP2-76393, Novus Biologicals; 1:1000), GFP (sc-9996, Santa Cruz Biotechnology; 1:1666), alpha-tubulin (NB100-690, Novus Biologicals; 1:5000), IRDye 680LT Goat anti-Rabbit (LI-COR 926–68021, 1:10,000), IRDye 800CW Goat anti-Mouse (LI-COR 926–32210, 1:10,000).

Techniques: Virus, Recombinant, Software

Journal: Circulation

Article Title: BRD4 (Bromodomain-Containing Protein 4) Interacts with GATA4 (GATA Binding Protein 4) to Govern Mitochondrial Homeostasis in Adult Cardiomyocytes

doi: 10.1161/circulationaha.120.047753

Figure Lengend Snippet: Figure 2. BRD4 regulates mitochondrial metabolic pathways in adult cardiomyocytes.

Article Snippet: Antibodies used for immunohistochemistry were: Brd4 (rabbit, Bethyl 00396) and Cleaved caspase 3 (rabbit, Cell Signaling 9664).

Techniques:

Journal: Circulation

Article Title: BRD4 (Bromodomain-Containing Protein 4) Interacts with GATA4 (GATA Binding Protein 4) to Govern Mitochondrial Homeostasis in Adult Cardiomyocytes

doi: 10.1161/circulationaha.120.047753

Figure Lengend Snippet: Figure 3. Constitutive cardiomyocyte-specific Brd4 deletion is embryonic lethal and reveals

Article Snippet: Antibodies used for immunohistochemistry were: Brd4 (rabbit, Bethyl 00396) and Cleaved caspase 3 (rabbit, Cell Signaling 9664).

Techniques:

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

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: 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.

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

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: 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.

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

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: 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.

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

Journal: Nature Communications

Article Title: BRD4 promotes resection and homology-directed repair of DNA double-strand breaks

doi: 10.1038/s41467-022-30787-6

Figure Lengend Snippet: a pDSB was replicated with α 32 P[dATP] in reactions containing buffer or JQ1 (BETi). After 45 min, reactions were supplemented with AgeI and samples were withdrawn for 1D gel electrophoresis ( n = 4 independent experiments). b , c Quantitation of linear ( b ) and HMW ( c ) molecules from ( a ). d Protein samples from ( a ) were withdrawn and analyzed by Western blot ( n = 2 independent experiments). e , f pDSB was replicated in extract containing buffer or JQ1 (BETi). After 45 min, reactions were supplemented with AgeI. Samples were withdrawn 30 min after enzyme addition and analyzed by amplicon sequencing ( n = 2 independent experiments). Results are graphed to show the frequency of insertion products ( e ), and the frequency of different deletion products ( f ). g pDSB was replicated in extract supplemented with buffer or JQ1 (BETi). After 45 min, AgeI was added and DNA-bound proteins were isolated by plasmid pull-down. Samples were analyzed by Western blot with the indicated antibodies ( n = 2 independent experiments). Non-specific band (*). h pDSB and a control plasmid lacking AgeI sites were replicated in extract. After 45 min, AgeI was added and samples were withdrawn 5 min later for analysis by BRD4 ChIP ( n = 3 independent experiments). i Schematic of undigested 2D gel intermediates. The relative position of open circular (OC), supercoiled (SC), and linear plasmids is indicated. An example of resected linear molecules is also shown. j pDSB was replicated with α 32 P[dATP] in reactions containing buffer or BETi. After 45 min, AgeI was added and samples were withdrawn 30 min later for 2D gel electrophoresis ( n = 2 independent experiments). k Quantitation of linear and resected molecules in ( j ). Arbitrary units (a.u.). Error bars represent ± one standard deviation from the mean. Student’s two-tailed t test: not significant (ns), p -value < 0.05 (*), p -value < 0.01 (**).

Article Snippet: Xenopus laevis BRD2, BRD3, and BRD4 antibodies were produced by New England Peptide (NEP) using the following antigen sequences: BRD2-KPHDKAESAHQVSVT, BRD3-EPRRERYKGATQAS, and BRD4-NFQSELMEIFEQNLFS (1:4000 dilution).

Techniques: Nucleic Acid Electrophoresis, Quantitation Assay, Western Blot, Amplification, Sequencing, Isolation, Plasmid Preparation, Control, Two-Dimensional Gel Electrophoresis, Electrophoresis, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: BRD4 promotes resection and homology-directed repair of DNA double-strand breaks

doi: 10.1038/s41467-022-30787-6

Figure Lengend Snippet: a Mock-depleted (ΔMock) or BRD4-depleted (ΔBRD4) NPE was analyzed by Western blot using the indicated antibodies ( n = 2 independent experiments). b pDSB was replicated with α 32 P[dATP] in mock- or BRD4-depleted extracts. After 60 min, reactions were supplemented with AgeI and samples were isolated at the indicated time points for 1D gel electrophoresis ( n = 2 independent experiments). c , d Quantitation of linear ( c ) and HMW ( d ) molecules from ( b ). e pDSB was replicated in mock- or BRD4-depleted extract. After 60 min, reactions were supplemented with AgeI and DNA-bound proteins were isolated by plasmid pull-down. Samples were analyzed by Western blot with the indicated antibodies ( n = 2 independent experiments). f A mixture of HSS and NPE was immunoprecipitated with beads conjugated to mock, BRD4, BRG1, or CtIP antibodies. Bead-bound proteins were analyzed by Western blot with the indicated antibodies ( n = 3 independent experiments). g Schematic showing interactions between BRD4-BRG1-CtIP and their role in coordinating DSB repair through nucleosome eviction, DNA end resection, and homologous recombination. Acetylation (Ac); Mre11-Rad50-Nbs1 (MRN).

Article Snippet: Xenopus laevis BRD2, BRD3, and BRD4 antibodies were produced by New England Peptide (NEP) using the following antigen sequences: BRD2-KPHDKAESAHQVSVT, BRD3-EPRRERYKGATQAS, and BRD4-NFQSELMEIFEQNLFS (1:4000 dilution).

Techniques: Western Blot, Isolation, Nucleic Acid Electrophoresis, Quantitation Assay, Plasmid Preparation, Immunoprecipitation, Homologous Recombination