flag tagged brd4 plasmid  (Mirus Bio)

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 1. BRD4 NCC loss of function produces severe craniofacial phenotypes.

Article Snippet: HEK293T cells were cotransfected with HA tagged human RUNX2 was modified from the Harvard plasmid database (HsCD00462359) and Flag tagged human BRD4 (addgene 90331).

Techniques:

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 2. BRD4 mutant mandibular cNCCs fail to properly differentiate to osteoblast lineages.

Article Snippet: HEK293T cells were cotransfected with HA tagged human RUNX2 was modified from the Harvard plasmid database (HsCD00462359) and Flag tagged human BRD4 (addgene 90331).

Techniques: Mutagenesis

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 3. Loss of BRD4 disrupts in vitro cNCCs osteoblast differentiation.

Article Snippet: HEK293T cells were cotransfected with HA tagged human RUNX2 was modified from the Harvard plasmid database (HsCD00462359) and Flag tagged human BRD4 (addgene 90331).

Techniques: In Vitro

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 4. BRD4 binds to proximal active enhancers to regulate osteogenic transcription.

Article Snippet: HEK293T cells were cotransfected with HA tagged human RUNX2 was modified from the Harvard plasmid database (HsCD00462359) and Flag tagged human BRD4 (addgene 90331).

Techniques:

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 5. BRD4 directly regulates transcription of factors critical for osteoblast differentiation.

Article Snippet: HEK293T cells were cotransfected with HA tagged human RUNX2 was modified from the Harvard plasmid database (HsCD00462359) and Flag tagged human BRD4 (addgene 90331).

Techniques:

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 6. BRD4 associates with RUNX2 to regulate osteoblast differentiation

Article Snippet: HEK293T cells were cotransfected with HA tagged human RUNX2 was modified from the Harvard plasmid database (HsCD00462359) and Flag tagged human BRD4 (addgene 90331).

Techniques:

Journal: Development (Cambridge, England)

Article Title: BRD4 binds to active cranial neural crest enhancers to regulate RUNX2 activity during osteoblast differentiation.

doi: 10.1242/dev.202110

Figure Lengend Snippet: Fig. 7. Model of BRD4 function in CdLS craniofacial pathogenesis (created with

Article Snippet: HEK293T cells were cotransfected with HA tagged human RUNX2 was modified from the Harvard plasmid database (HsCD00462359) and Flag tagged human BRD4 (addgene 90331).

Techniques:

Journal: PLoS Pathogens

Article Title: BRD4 inhibition exerts anti-viral activity through DNA damage-dependent innate immune responses

doi: 10.1371/journal.ppat.1008429

Figure Lengend Snippet: (a) A list of ten epigenetic agents. (b) Schematic for GFP-reporter assays. PK15 cells were seeded in 24-well plates. After the cells were cultured to approximately 60%–70% confluence, the compounds were added to cells for 4 h at 37°C. Then cells were incubated with PRV-GFP (MOI = 0.01) and different concentrations of compounds for another 36 h at 37°C. Flow cytometry analysis was carried out to examine the GFP fluorescence representing viral replication. (c-l) Results of flow cytometry assays with small-molecule inhibitors listed in a. Data are shown as mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t- test. (m) BRD4 was assessed with immunoblotting in Scramble, shBRD4-1 and shBRD4-2 PK15 cells. Actin served as a loading control. (n) PRV-GFP proliferation was assessed by flow cytometry in Scramble, shBRD4-1 and shBRD4-2 PK15 cells infected with PRV-GFP (MOI = 0.01) and treated with DMSO, JQ-1 (1 μM), OTX-015 (10 μM) and I-BET 151 (10 μM) for 36 h. Data are shown as mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by one-way ANOVA. ns, no significance.

Article Snippet: The plasmid for expression of flag-tagged BRD4 that was resistant to BRD4 shRNAs was synthesized and constructed by GenScript.

Techniques: Cell Culture, Incubation, Flow Cytometry, Fluorescence, Two Tailed Test, Western Blot, Control, Infection

Journal: PLoS Pathogens

Article Title: BRD4 inhibition exerts anti-viral activity through DNA damage-dependent innate immune responses

doi: 10.1371/journal.ppat.1008429

Figure Lengend Snippet: (a) c-Myc and c-Jun mRNA were assessed with RT-qPCR analysis in PK15 cells treated with JQ-1 (1 μM) at 0–36 hpt. Data are shown as mean ± SD based on three independent experiments. *** P < 0.001 determined by two-tailed Student’s t -test. (b) IFN-β and BRD4 mRNA were assessed with RT-qPCR analysis in PK15 cells infected with PRV-QXX (MOI = 0.1) at 0–24 hpi. Data are shown as mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test. (c) BRD4 expression was assessed with immunoblotting in PK15 cells infected with PRV-QXX (MOI = 0.1) at 0–24 hpi. Actin served as a loading control. (d-f) PRV UL9 (d), UL5 (e) and US1 (f) mRNA were assessed with RT-qPCR analysis in PK15 cells infected with PRV-QXX (MOI = 0.1) and treated with DMSO or JQ-1 (1 μM) at 0–24 hpt. (g) PRRSV ORF7 mRNA were assessed with RT-qPCR analysis in MARC-145 cells infected with PRRSV-BJ4 (MOI = 1) and treated with DMSO or JQ-1 (1 μM) at 0–36 hpt.

Article Snippet: The plasmid for expression of flag-tagged BRD4 that was resistant to BRD4 shRNAs was synthesized and constructed by GenScript.

Techniques: Quantitative RT-PCR, Two Tailed Test, Infection, Expressing, Western Blot, Control

Journal: PLoS Pathogens

Article Title: BRD4 inhibition exerts anti-viral activity through DNA damage-dependent innate immune responses

doi: 10.1371/journal.ppat.1008429

Figure Lengend Snippet: (a and b) Viral attachment was assessed with RT-qPCR analysis in PK15 cells incubated with PRV-QXX (a, MOI = 1) or VSV-GFP (b, MOI = 0.01). (c) Viral attachment was assessed with RT-qPCR analysis in MARC-145 cells incubated with PRRSV-BJ4 (MOI = 10). (d) Viral attachment was assessed with immunoblotting analysis against PRV gE in PK15 cells incubated with PRV-QXX (MOI = 1). Actin served as a loading control. (e) Viral attachment was assessed with fluorescence analysis in PK15 cells incubated with EdU-labeled PRV-QXX (MOI = 1) which were detected by Apollo staining (red). CellMask Green staining (green) indicated the plasma membrane. Scale bar, 10 μM. (f) Viral attachment was assessed with fluorescence analysis in Vero cells incubated with EdU-labeled ECTV (MOI = 10) which were detected by Apollo staining (green). CellMask Deep red staining (red) indicated the plasma membrane. Scale bar, 10 μM. (g) BRD4 expression was assessed with immunoblotting in Scramble, shBRD4-1, shBRD4-2, shBRD4-1/BRD4 and shBRD4-2/BRD4 PK15 cells. Actin served as a loading control. (h and i) Viral attachment was assessed with RT-qPCR analysis in Scramble, shBRD4-1, shBRD4-2, shBRD4-1/BRD4 and shBRD4-2/BRD4 PK15 cells incubated with PRV-QXX (h, MOI = 1) or VSV-GFP (i, MOI = 0.01). All data are shown as mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t -test.

Article Snippet: The plasmid for expression of flag-tagged BRD4 that was resistant to BRD4 shRNAs was synthesized and constructed by GenScript.

Techniques: Quantitative RT-PCR, Incubation, Western Blot, Control, Fluorescence, Labeling, Staining, Clinical Proteomics, Membrane, Expressing, Two Tailed Test

Journal: PLoS Pathogens

Article Title: BRD4 inhibition exerts anti-viral activity through DNA damage-dependent innate immune responses

doi: 10.1371/journal.ppat.1008429

Figure Lengend Snippet: BRD4 inhibition leads to DDR-induced activation of cGAS-mediated innate immune pathway, which exhibits broadly antiviral activity through impairment of viral attachment.

Article Snippet: The plasmid for expression of flag-tagged BRD4 that was resistant to BRD4 shRNAs was synthesized and constructed by GenScript.

Techniques: Inhibition, Activation Assay, Activity Assay

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Schematic diagram depicting different types of alternatively spliced events (left). The bar graph (right) shows the distribution of alternatively spliced events among those that are differentially spliced in total thymus in BRD4 knock-out versus wild type (WT) (FDR < 0.05). Comparison of splicing events between WT and BRD4-deficient cells was based on transcripts expressed in both. (B) Developmental stages in thymocyte differentiation, DN (CD4, CD8 DN), ISP (CD8+ ISP), DP (CD4, CD8 DP), CD4, and CD8 single-positive thymocytes are shown. Arched arrows denote level of proliferative activity in DN and ISP thymocytes. (C) Bar graph showing the total number of differentially spliced events in the different thymocyte subpopulations in BRD4 knock-out versus wild-type thymus (FDR < 0.05), derived from RNA-seq analysis. BRD4 was conditionally deleted in DN thymocytes by LCK-Cre (Gegonne et al., 2018). Comparison of splicing events between WT and BRD4-deficient cells was based on transcripts expressed in both. See also Figure S1.

Article Snippet: Human Flag-tagged BRD4 ΔET in pCMV2 mammalian vector , ADDgene ( Bisgrove et al., 2007 ) , #21938; RRID: Addgene_21938.

Techniques: Knock-Out, Comparison, Activity Assay, Derivative Assay, RNA Sequencing

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: Summary of Immune Relevant Genes Whose Patterns of Splicing Are Altered by BRD4 Deficiency and the Thymocyte Subset in which the Alteration Occurs

Article Snippet: Human Flag-tagged BRD4 ΔET in pCMV2 mammalian vector , ADDgene ( Bisgrove et al., 2007 ) , #21938; RRID: Addgene_21938.

Techniques: Binding Assay

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: RNA from the different thymocyte subpopulations was subjected to RT-PCR for the indicated genes: CD45 (A and B), Arhgef1 (C and D), and Picalm (E and F). (A, C, and E) Upper panels: schematic diagrams depicting partial gene structure of the alternatively spliced genes CD45 (A), Arhgef1 (C), and Picalm (E). Rectangular boxes represent the exons, and the horizontal straight lines connecting the boxes represent the introns; the numbers below the boxes refer to the exon number of the gene, and numbers inside the boxes refer to the length of the exons; the numbers within the terminal exons do not refer to the actual exon length but the length amplifiable by the RT-PCR primers. The arrow heads show the approximate positions of the RT-PCR primers; boxes with hashed lines show the alternative exons; and curved lines connecting the boxes depict the splicing pattern. WT and KO refer to the splicing pattern prevalent in either the wild-type or knock-out thymocytes as determined by RNA-seq analysis. Lower panels: ethidium bromide stained agarose gels showing RT-PCR products derived from total RNA from BRD4 WT and KO thymocytes. (B, D, and F) Bar graphs of the RT-PCR results for CD45 (B), Arhgef1 (D), and Picalm (F). The ratios A/A+B (ratio of included exon transcript/total transcripts) were used as measure of alternative splicing and represent the average of three separate RT-PCR analyses. #, p < 0.05, significant difference between WT subpopulations, relative to WT DN; *p < 0.05, significant difference between WT and KO for the specific subpopulation. See also Figure S2.

Article Snippet: Human Flag-tagged BRD4 ΔET in pCMV2 mammalian vector , ADDgene ( Bisgrove et al., 2007 ) , #21938; RRID: Addgene_21938.

Techniques: Reverse Transcription Polymerase Chain Reaction, Knock-Out, RNA Sequencing, Staining, Derivative Assay, Alternative Splicing

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Effect of JQ1 or dBET6 treatment on the binding of BRD4 across the gene. ALL, TSS+gene body+TTS+intergenic; TSS, transcription start site; Gene body, between TSS and TTS; TTS, transcription termination site; intergenic, all remaining sequences. The peak distribution, in the absence of treatment is as follows: TSS, 1123; gene body, 4065; TTS, 226; intergenic, 1827. (B) Bar graph showing the distribution of alternative splice events among the differentially spliced events in response to JQ1 treatment or dBET6 treatment in T-ALL cells. (C) Bar graph showing the fraction of alternative splice (AS) genes that also have BRD4 associated with them at the TSS (pkAS). The total number of BRD4 peaks detected at the TSS across the genome was 1123. (D) Bar graph showing the fraction of AS genes that are also differentially expressed (DE) in response to JQ1 or dBET6 treatment. p values for (C) and (D) were obtained using a hypergeometric test, which tests the probability that the frequency of AS genes derived from either DE genes (overlap) or genes with BRD4-bound TSS peaks is larger than expected from the population; a low p value suggests the enrichment of AS genes in either DE genes or genes with BRD4 TSS peaks. See also Figures S3 and S4.

Article Snippet: Human Flag-tagged BRD4 ΔET in pCMV2 mammalian vector , ADDgene ( Bisgrove et al., 2007 ) , #21938; RRID: Addgene_21938.

Techniques: Binding Assay, Derivative Assay

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Immunoblot of BRD4 immunoprecipitates from thymocyte nuclear extracts (with and without benzonase treatment) with indicated antibodies to splicing factors FUS, HnRNPL, and U1–70. The immunoprecipitates from a single extract were run on either a 6% gel to visualize BRD4 and Fus or on a 10% gel to visualize HnRNPL and U1–70. The values under the IP lanes indicate the enrichment of anti-BRD4 co-IP, relative to the IgG control. (B, left) Immunoblot of BRD4 immunoprecipitates from HeLa nuclear extracts with indicated antibodies to splicing factors FUS, HnRNPM, U1–70, and U1-A. (B, right) Immunoblot of FUS immunoprecipitates from HeLa nuclear extracts with indicated antibodies to BRD4 and splicing factors HnRNPM, U1–70, and U1-A. (C) Schematic representation of BRD4 and BRD4-deletion mutants. The coordinates of the mouse BRD4 mutations are as follows. WT BRD4, 1402 aa; DN, 722–1402 aa; ΔC, 1–699aa; ΔBD1, 146–1402 aa; ΔBD2+B, 1–349/599–1402 aa; ΔB, 1–502/549–1402 aa; ΔET, 1–600/684–1402 aa; ΔHAT, 1–1156/1198–1402 aa. (D) Immunoblots showing pull-down analysis of recombinant BRD4 with recombinant HnRNPM. rHnRNPM (0.25 μg) was pulled down with rflag-BRD4 (0.5 μg) immobilized on Flag beads. Immunoblots were with anti-HnRNPM (upper) and anti-BRD4 (lower). (E) Immunoblots showing pull-down analysis of recombinant BRD4 with recombinant FUS. rFUS (0.25 μg) was pulled down with rflag-BRD4 (0.5 μg) WT or equimolar amounts of N-terminal or C-terminal BRD4 truncation mutants immobilized on Flag beads. Immunoblots were with anti-FUS (upper) and anti-BRD4 (lower). (F) Binding of HnRNPM (left panel) and FUS (right panel) to BRD4 mutants was assessed in pull-down assays with rBRD4 immobilized on Flag beads and immunoblotting with appropriate antibodies. The results represent the average of two experiments. (G) Retention of FUS and HnRNPM to BRD4 mutants, relative to the WT, was quantified as the fraction of input and normalized to the extent of binding to BRD4 WT. All results are representative of at least two independent experiments. See also Figure S5.

Article Snippet: Human Flag-tagged BRD4 ΔET in pCMV2 mammalian vector , ADDgene ( Bisgrove et al., 2007 ) , #21938; RRID: Addgene_21938.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Control, Recombinant, Binding Assay

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Proximity ligation assays (PLAs) were performed on primary thymocytes with anti-BRD4 and the antibodies for the indicated splicing factors. The PLAs are all significantly above the single antibody controls (Figure S6C). (B) PLA was performed using anti-BRD4 and the antibodies for the indicated splicing factors on fixed HeLa cells that had been treated with JQ1 (500 nM)/ DMSO for 6 hr. There is no significant difference (p > 0.05) between the treated and control PLA samples for either HnRNPM or Fus; both PLAs are significantly above single antibody alone controls (Figure S6C). PLA interaction is shown in red; DAPI staining in blue. See also Figures S6C and S6D.

Article Snippet: Human Flag-tagged BRD4 ΔET in pCMV2 mammalian vector , ADDgene ( Bisgrove et al., 2007 ) , #21938; RRID: Addgene_21938.

Techniques: Ligation, Control, Staining

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: (A) Metagene profile of BRD4 and FUS CHIP datasets showing colocalization of BRD4 and FUS at the TSS. (B) Log2 enrichment of reads in genomic features along the metagene body. (C) Enrichment heatmap showing co-localization of BRD4 with FUS across the genome. (D) Genome browser views of DNAAF3, ROBO3, and MAN1A1, showing BRD4 and FUS co-localization around the TSS and gene body. See also Figures S6A and S6B.

Article Snippet: Human Flag-tagged BRD4 ΔET in pCMV2 mammalian vector , ADDgene ( Bisgrove et al., 2007 ) , #21938; RRID: Addgene_21938.

Techniques:

Journal: Cell reports

Article Title: The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

doi: 10.1016/j.celrep.2019.10.066

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human Flag-tagged BRD4 ΔET in pCMV2 mammalian vector , ADDgene ( Bisgrove et al., 2007 ) , #21938; RRID: Addgene_21938.

Techniques: Recombinant, In Situ, cDNA Synthesis, Plasmid Preparation, Mutagenesis, Clone Assay, Software, Sequencing, Alternative Splicing