anti ebf 1  (Novus Biologicals)

Journal: Development (Cambridge, England)

Article Title: The transcription factor EBF1 non-cell-autonomously regulates cardiac growth and differentiation.

doi: 10.1242/dev.202054

Figure Lengend Snippet: Fig. 1. Abnormal cardiac form in Ebf1 knockout mice. (A) Ebf1 wild-type and knockout mice at postnatal day 21. (B) Hematoxylin and Eosin- stained images of postnatal day 21 wild-type and KO heart sections. (C) Heart weight to body weight ratio measurements in wild-type and KO mice. (D) Echocardiographic measurements of the left ventricular wall thickness (IVSd, interventricular septum diastole; PWd, posterior wall diastole), left ventricular end diastolic diameter (LVEDD), fractional shortening (FS) and relative wall thickness (RWT) in Ebf1 wild-type and KO mice. Scale bars: 1 cm in A; 1 mm in B. *P<0.05 (unpaired t-test). WT, wild type.

Article Snippet: Primary antibodies were directed against EBF1 (R&D Systems; AF5165; 1:50), endomucin (Abcam; AB106100; 1:200), α-actinin (Abcam AB9465; 1:00), troponin T (Fisher Scientific; BD B564766; 1:100), hyaluronic acid binding protein biotinylated (MilliporeSigma; 38591150UG, 1:200), Ki-67 (Abcam; AB16667; 1:100), ERG (Abcam; ab92513; 1:100), c-MYC (Abcam; AB32072; 1:100), GFP (Abcam; AB13970; 1:100), WGA (Thermo Fisher Scientific; W112621; 1:500) and visualized by confocal microscopy (Leica SP5).

Techniques: Knock-Out, Staining

Journal: Development (Cambridge, England)

Article Title: The transcription factor EBF1 non-cell-autonomously regulates cardiac growth and differentiation.

doi: 10.1242/dev.202054

Figure Lengend Snippet: Fig. 2. Myocardial hyperplasia in Ebf1 knockout mice. (A) High- magnification Hematoxylin and Eosin-stained images of the left ventricular free wall with cardiomyocytes in short axis (upper panels) and long axis (lower panels). (B) Quantitative image analysis of nuclei per high power field in Ebf1 wild-type and KO cardiomyocytes. (C) Immunofluorescence staining of wheat germ agglutinin (WGA), α-actinin and DAPI. (D) Violin plot of cell size distribution in Ebf1 wild-type and KO hearts. Each violin represents a separate heart. Scale bars: 20 mm. *P<0.05, ****P<0.001 (unpaired t-test). WT, wild type.

Article Snippet: Primary antibodies were directed against EBF1 (R&D Systems; AF5165; 1:50), endomucin (Abcam; AB106100; 1:200), α-actinin (Abcam AB9465; 1:00), troponin T (Fisher Scientific; BD B564766; 1:100), hyaluronic acid binding protein biotinylated (MilliporeSigma; 38591150UG, 1:200), Ki-67 (Abcam; AB16667; 1:100), ERG (Abcam; ab92513; 1:100), c-MYC (Abcam; AB32072; 1:100), GFP (Abcam; AB13970; 1:100), WGA (Thermo Fisher Scientific; W112621; 1:500) and visualized by confocal microscopy (Leica SP5).

Techniques: Knock-Out, Staining, Immunofluorescence

Journal: Development (Cambridge, England)

Article Title: The transcription factor EBF1 non-cell-autonomously regulates cardiac growth and differentiation.

doi: 10.1242/dev.202054

Figure Lengend Snippet: Fig. 3. Persistent postnatal cardiomyocyte proliferation in Ebf1 knockout mice. (A) Immunostaining of heart sections from P21 wild-type and knockout mice for KI-67, TNNT2 and DAPI. (B) Immunostaining of heart sections from P21 wild-type and two separate knockout mice for KI-67, NKX2.5 and DAPI. Lower panels show high-magnification images of the areas outlined in the top panels. White arrowheads indicate nuclei positive for both Ki-67 and NKX2.5. Yellow arrowheads indicate nuclei positive for Ki-67 only. Scale bar: 25 µm. WT, wild type.

Article Snippet: Primary antibodies were directed against EBF1 (R&D Systems; AF5165; 1:50), endomucin (Abcam; AB106100; 1:200), α-actinin (Abcam AB9465; 1:00), troponin T (Fisher Scientific; BD B564766; 1:100), hyaluronic acid binding protein biotinylated (MilliporeSigma; 38591150UG, 1:200), Ki-67 (Abcam; AB16667; 1:100), ERG (Abcam; ab92513; 1:100), c-MYC (Abcam; AB32072; 1:100), GFP (Abcam; AB13970; 1:100), WGA (Thermo Fisher Scientific; W112621; 1:500) and visualized by confocal microscopy (Leica SP5).

Techniques: Knock-Out, Immunostaining

Journal: Development (Cambridge, England)

Article Title: The transcription factor EBF1 non-cell-autonomously regulates cardiac growth and differentiation.

doi: 10.1242/dev.202054

Figure Lengend Snippet: Fig. 4. Abnormal ventricular conduction system form and function in Ebf1-null mice. (A) ECG tracings from wild-type and Ebf1 KO animals shows QRS duration prolongation. Red lines indicate the beginning and end of the QRS complex. Scale bar: 50 ms. (B) ECG intervals in wild-type and Ebf1 KO animals. QRS and QT intervals are significantly prolonged. (C) Optical maps of the anterior epicardial surface show abnormal patterns of ventricular depolarization in KO animals. (D) Bar graph of conduction velocity and action potential duration at 50% repolarization (APD50) measured from activation maps of wild-type and Ebf1 KO animals. (E) Whole-mount images of left ventricular endocardial surface of wild-type and Ebf1 KO mice using the Cntn2-EGFP reporter. (F,G) Quantitative image analysis of VCS area as a percentage of left ventricular endocardial surface (F) and FACS analysis of the proportion of GFP+ myocytes (G) from postnatal day 21 Ebf1 wild-type and KO hearts in Cntn2-EGFP background. Scale bars: 1 mm. **P<0.01, ***P<0.001, ****P<0.0001 (unpaired t-test). WT, wild type.

Article Snippet: Primary antibodies were directed against EBF1 (R&D Systems; AF5165; 1:50), endomucin (Abcam; AB106100; 1:200), α-actinin (Abcam AB9465; 1:00), troponin T (Fisher Scientific; BD B564766; 1:100), hyaluronic acid binding protein biotinylated (MilliporeSigma; 38591150UG, 1:200), Ki-67 (Abcam; AB16667; 1:100), ERG (Abcam; ab92513; 1:100), c-MYC (Abcam; AB32072; 1:100), GFP (Abcam; AB13970; 1:100), WGA (Thermo Fisher Scientific; W112621; 1:500) and visualized by confocal microscopy (Leica SP5).

Techniques: Activation Assay

Journal: Development (Cambridge, England)

Article Title: The transcription factor EBF1 non-cell-autonomously regulates cardiac growth and differentiation.

doi: 10.1242/dev.202054

Figure Lengend Snippet: Fig. 5. EBF1 regulates myocardial architecture and proliferation during heart development. (A) Hematoxylin and Eosin staining of transmural sections from the left ventricles of E13.5 wild-type and Ebf1 mutant hearts at the midventricular level. (B) Expanded views of areas outlined in A. Red arrowhead indicates extracellular matrix bubble. (C) Immunostaining of the extracellular matrix using hyaluronic acid-binding protein (HABP) and of cardiomyocytes using troponin T2 (TNNT2) in wild-type and Ebf1 mutant hearts. (D) Quantification of the ratio of HABP+ area to total myocardial area in wild-type and Ebf1 mutant hearts. (E) Immunostaining for the Ki-67 proliferation marker along with the endocardial marker endomucin (EMCN) in wild-type and Ebf1 mutant hearts. (F) Quantification of the ratio of Ki-67+ nuclei to total nuclei in wild-type and Ebf1 mutant hearts. Scale bars: 25 µm in A,C,E; 10 μm in B. *P<0.05, **P<0.01 (unpaired t-test). WT, wild type.

Article Snippet: Primary antibodies were directed against EBF1 (R&D Systems; AF5165; 1:50), endomucin (Abcam; AB106100; 1:200), α-actinin (Abcam AB9465; 1:00), troponin T (Fisher Scientific; BD B564766; 1:100), hyaluronic acid binding protein biotinylated (MilliporeSigma; 38591150UG, 1:200), Ki-67 (Abcam; AB16667; 1:100), ERG (Abcam; ab92513; 1:100), c-MYC (Abcam; AB32072; 1:100), GFP (Abcam; AB13970; 1:100), WGA (Thermo Fisher Scientific; W112621; 1:500) and visualized by confocal microscopy (Leica SP5).

Techniques: Staining, Mutagenesis, Immunostaining, Binding Assay, Marker

Journal: Development (Cambridge, England)

Article Title: The transcription factor EBF1 non-cell-autonomously regulates cardiac growth and differentiation.

doi: 10.1242/dev.202054

Figure Lengend Snippet: Fig. 6. EBF1 expression in the murine heart. (A,B) Immunofluorescence of EBF1 co-stained with EMCN (A) or the myocyte marker α-actinin (B) in E13.5 embryonic heart sections. (C-E) Immunofluorescence of EBF1 co-stained with the endothelial marker ERG and the fibroblast/smooth muscle cell marker PDGFRα in heart sections at (C) E13.5, (D) postnatal day 1 and (E) postnatal day 21. High- magnification images of areas outlined in E are shown underneath. All images are at the mid level of the left ventricle. Scale bars: 25 µm.

Article Snippet: Primary antibodies were directed against EBF1 (R&D Systems; AF5165; 1:50), endomucin (Abcam; AB106100; 1:200), α-actinin (Abcam AB9465; 1:00), troponin T (Fisher Scientific; BD B564766; 1:100), hyaluronic acid binding protein biotinylated (MilliporeSigma; 38591150UG, 1:200), Ki-67 (Abcam; AB16667; 1:100), ERG (Abcam; ab92513; 1:100), c-MYC (Abcam; AB32072; 1:100), GFP (Abcam; AB13970; 1:100), WGA (Thermo Fisher Scientific; W112621; 1:500) and visualized by confocal microscopy (Leica SP5).

Techniques: Expressing, Immunofluorescence, Staining, Marker

Journal: Development (Cambridge, England)

Article Title: The transcription factor EBF1 non-cell-autonomously regulates cardiac growth and differentiation.

doi: 10.1242/dev.202054

Figure Lengend Snippet: Fig. 7. Gene profiling and chromatin analysis of embryonic Ebf1−/−non-myocytes. (A) Heatmap of the top 100 DEGs between E13.5 non-myocyte cells (NMCs) isolated from wild-type and KO hearts. (B) Volcano plot of DEGs. (C) Top 20 ChIP-Seq gene sets identified using ChEA3 analysis of DEGs. (D) Volcano plot of differentially accessible regions (DARs) identified by ATAC-Seq in wild- type and KO NMCs. (E) Top 20 ChIP-Seq gene sets identified using ChEA3 analysis of genes near DARs. (F) Venn diagram showing the genes shared between RNA-Seq identified DEGs and ATAC-Seq identified DARs in KO NMCs, and previously validated Ebf1 target genes. (G) Transcription factors shared between RNA-Seq, ATAC-Seq and ChIP-Seq analyses, along with relative expression in knockout cells and statistical significance.

Article Snippet: Primary antibodies were directed against EBF1 (R&D Systems; AF5165; 1:50), endomucin (Abcam; AB106100; 1:200), α-actinin (Abcam AB9465; 1:00), troponin T (Fisher Scientific; BD B564766; 1:100), hyaluronic acid binding protein biotinylated (MilliporeSigma; 38591150UG, 1:200), Ki-67 (Abcam; AB16667; 1:100), ERG (Abcam; ab92513; 1:100), c-MYC (Abcam; AB32072; 1:100), GFP (Abcam; AB13970; 1:100), WGA (Thermo Fisher Scientific; W112621; 1:500) and visualized by confocal microscopy (Leica SP5).

Techniques: Isolation, ChIP-sequencing, RNA Sequencing, Expressing, Knock-Out

Journal: Development (Cambridge, England)

Article Title: The transcription factor EBF1 non-cell-autonomously regulates cardiac growth and differentiation.

doi: 10.1242/dev.202054

Figure Lengend Snippet: Fig. 8. MYC overexpression in embryonic Ebf1−/−

Article Snippet: Primary antibodies were directed against EBF1 (R&D Systems; AF5165; 1:50), endomucin (Abcam; AB106100; 1:200), α-actinin (Abcam AB9465; 1:00), troponin T (Fisher Scientific; BD B564766; 1:100), hyaluronic acid binding protein biotinylated (MilliporeSigma; 38591150UG, 1:200), Ki-67 (Abcam; AB16667; 1:100), ERG (Abcam; ab92513; 1:100), c-MYC (Abcam; AB32072; 1:100), GFP (Abcam; AB13970; 1:100), WGA (Thermo Fisher Scientific; W112621; 1:500) and visualized by confocal microscopy (Leica SP5).

Techniques: Over Expression

Journal: Development (Cambridge, England)

Article Title: The transcription factor EBF1 non-cell-autonomously regulates cardiac growth and differentiation.

doi: 10.1242/dev.202054

Figure Lengend Snippet: Fig. 9. BMP-mediated regulation of Ebf1 expression in embryonic hearts. (A) Bar graph of Ebf1 expression in E9.5 heart cultures exposed to various ligands for 48 h. (B) Bar graph of Ebf1 expression in E9.5 heart cultures exposed to control vehicle, BMP10, LDN or gremlin 2 for 48 h. (C) Volcano plot of DEGs in embryonic heart cultures exposed to LDN for 48 h. (D) Venn diagram of DEGs of LDN-treated hearts and EBF1 gene targets using published ChIP-Seq data (Ungerbä ck et al., 2015) from pro B-cells. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (one way ANOVA).

Article Snippet: Primary antibodies were directed against EBF1 (R&D Systems; AF5165; 1:50), endomucin (Abcam; AB106100; 1:200), α-actinin (Abcam AB9465; 1:00), troponin T (Fisher Scientific; BD B564766; 1:100), hyaluronic acid binding protein biotinylated (MilliporeSigma; 38591150UG, 1:200), Ki-67 (Abcam; AB16667; 1:100), ERG (Abcam; ab92513; 1:100), c-MYC (Abcam; AB32072; 1:100), GFP (Abcam; AB13970; 1:100), WGA (Thermo Fisher Scientific; W112621; 1:500) and visualized by confocal microscopy (Leica SP5).

Techniques: Expressing, Control, ChIP-sequencing

Journal: PLoS Pathogens

Article Title: Reduced IRF4 expression promotes lytic phenotype in Type 2 EBV-infected B cells

doi: 10.1371/journal.ppat.1010453

Figure Lengend Snippet: Immunoblot analyses were performed to compare the protein expression levels of IRF4 (A) or EBF1 and Z (B) in T1 versus T2 LCLs as indicated (using the same cell lysates). Tubulin was used as loading control. The numbers below the IRF4 immunoblot quantify the results using Image Studio Lite software to normalize the level of IRF4 expression to tubulin expression. Results are presented as the ratio of IRF4 expression relative to tubulin, in T2 cells (averaged) relative to T1 cells (averaged). The T1 IRF4 value is set as 1. The transcript expression of IRF4 (as determined by RNA-seq) is also shown for various different Type 1 and Type 2 LCL lines, along with the log2FC fold change in gene expression in T2 versus T1 cell lines, and the adjusted p value.

Article Snippet: The following antibodies were used for immunoblot analyses in this study: anti-R rabbit polyclonal antibody directed against the R peptide (peptide sequence EDPDEETSSQAVKALREMAD), anti-BZLF1 (Santa Cruz #sc-53904), anti-BMRF1 (Millipore #MAB8186), anti-IRF4 (Santa Cruz #sc-56713), anti-EBF1 (Biotechne #AF5165), anti-CD11C (Cell Signaling #45581), anti-caspase 1 (Abclonal #A0964), anti-ENPP2 (Proteintech #14243-1-AP), anti-Runx1 (Cell Signaling #4336S), anti-RUNX3 (Cell Signaling #13089), anti-NFATc1 (Santa Cruz #sc-7294), anti-NFATc2 (Cell Signaling #4389), anti-FYN (Santa Cruz #sc-434), anti-TNFRSF9 (Cell Signaling #34594), anti-CD9 (Cell Signaling #13174), anti-HLA DR/MHC class II (Santa Cruz #sc—53319), anti-AHR (Biotechne #AF6185), anti-CYP1B (Proteintech #18505-1-AP), anti-phospho-ERK (Cell Signaling #9101), anti-Tubulin (Sigma #T5168), anti-actin (Sigma #A5441), anti-V5 (Santa Cruz #sc—58052), and anti LMP2A (Santa Cruz #sc-101314).

Techniques: Western Blot, Expressing, Software, RNA Sequencing Assay