rabbit polyclonal anti-ebf1  (MyBiosource Biotechnology)

Journal: Histochemistry and Cell Biology

Article Title: EBF1 is expressed in pericytes and contributes to pericyte cell commitment

doi: 10.1007/s00418-021-02015-7

Figure Lengend Snippet: Primary antibodies used in the study

Article Snippet: Rabbit polyclonal anti-EBF1 (WB) , MyBioSource , MBS2027769.

Techniques:

Journal: Blood

Article Title: EBF1 and PAX5 control pro-B cell expansion via opposing regulation of the Myc gene

doi: 10.1182/blood.2020009564

Figure Lengend Snippet: Loss of functional EBF1 results in reduced levels of Myc transcripts and cell cycle arrest in pro-B cells. (A) Schematic drawing of the basic experimental layout. Neonatal Ebf1−/− BM or Ebf1−/− FL were transduced with EBF1-MIG (EBF1) or an EBF1-estrogen receptor fusion protein (EBF1-ER) construct. Cells were then exposed to 4-OHT to allow nuclear translocation of EBF1 in Ebf1-deficient cells carrying the EBF1-ER construct, which drives development into the CD19+ stage. 4-OHT was then withdrawn to test the dependency of EBF1 in the generated CD19+ cells. (B) Number of live cells of cultured EBF1 or EBF1-ER transduced and 4-OHT–treated Ebf1−/− BM cell cultures (as in panel A). Cells were either incubated continuously with 4-OHT (+4-OHT) or with 4-OHT withdrawn (-4-OHT) for 48 to 72 hours as indicated. Mean and standard deviation (SD) are shown; n = 4, from 4 culture experiments. (C) Cell cycle distributions of the cells in panel B at 48 hours after 4-OHT withdrawal. Mean and standard error of the mean are shown, n = 6). (D) The experimental protocol used to test the cell autonomous role of EBF1 in pro–B cell survival in vivo. Ebf1−/− FL cells were transduced to express EBF1 or EBF1-ER and treated with 4-OHT for 14 days. At day 15, a total of 1 million green fluorescent protein (GFP)+CD19+ cells were transplanted into C57BSJL (CD45.1) sublethally irradiated recipients. Donor reconstitution (CD45.2+GFP+), as well as CD19 expression, was determined by FACS 3 weeks posttransplantation. (E-F) Relative cell counts, from the BM of mice transplanted with either EBF1- or EBF1-ER–transduced cells. Mean and SD are shown; EBF1 n = 8, EBF1-ER n = 11, from 2 independent experiments. (G) Diagrams with quantitative reverse transcription PCR data from in vitro expanded Ebf1−/− FL pro-B control cells (FL) or BM cells from EBF1-deficient mice driven to B-cell progenitor stages with conventional or ER-fused EBF1 protein cultured in the presence or absence of 4-OHT for 72 hours. Mean and SD are shown; n = 4 to 7, from 2 independent experiments. For panels C and E-G, each dot indicates a data point, and the statistical analysis is based on a Student unpaired t test. *P < .05; **P < .01; ***P < .001; ****P < .0001.

Article Snippet: Ten μg per 10 7 cells of antibody rabbit anti-EBF1 polyclonal IgG (ABE1294; Millipore, Burlington, MA) was hybridized to 70 μl Protein-G Dynabeads (Life Technologies) and ChIP-seq was performed as described in the supplemental Materials and methods.

Techniques: Functional Assay, Transduction, Construct, Translocation Assay, Generated, Cell Culture, Incubation, Standard Deviation, In Vivo, Irradiation, Expressing, In Vitro

Journal: Blood

Article Title: EBF1 and PAX5 control pro-B cell expansion via opposing regulation of the Myc gene

doi: 10.1182/blood.2020009564

Figure Lengend Snippet: Ectopic expression of MYC rescues pro–B cell expansion in the absence of EBF1. Cell recovery from cultures of FL Ebf1−/− cells in vitro cultivated as described in Figure 1A. Cells were transduced with an empty MIG-GFP (MIGR1) vector (A), an EBF1-GFP vector (EBF1) (B), or an ER-fused EBF1 protein (EBF1-ER) (C) and serially transduced with an RFP control or a MYC-expressing RFP retrovirus. The diagrams display cell recovery when the cells were grown in the presence or absence of 4-OHT for 72 hours. Mean and standard deviation are shown; n = 6 to 12, from 3 independent experiments. (D) Recovery of CD45.2+CD19+GFP+RFP+ BM cells 3 to 4 weeks after transplantation of Ebf1−/− FL pro-B cells transduced with EBF1-ER and either a control RFP or MYC-expressing RFP-encoding retrovirus into sublethally irradiated CD45.1 mice. Mean and standard deviation are shown; n = 4 to 6 transplanted mice. (E) Representative FACS plot of immunoglobulin M (IgM) and CD19 expression on recipient (CD45.1) and donor (CD45.2+GFP+RFP+) cells. (F) Fraction of early (Annexin V–positive/4′6-diamidino-2-phenylindole–negative [red bar]) and late (Annexin V–positive/4′6-diamidino-2-phenylindole–positive [blue bar]) apoptotic cells 48 hours after removal of 4-OHT in EBF1- and EBF1-ER–expressing Ebf1−/− BM cells transduced with either RFP-control or RFP-Myc virus. Mean and standard error of the mean are shown; n = 3. The statistical analysis is based on comparisons of data from cells grown in the presence or absence of 4-OHT. (G) Histograms displaying overlays of an FACS staining of MYC protein in different stages of the cell cycle. (H) Bars depicting the ratios of MYC median fluorescent intensity (MFI) of each stage of the cell cycle compared with G0 48 hours after 4-OHT withdrawal in Wt and Ebf1−/−EBF1-ER BM cells Mean and standard error of the mean are shown, n = 6. The statistical analysis is based on a Student unpaired t test. *P < .05; **P < .01; ***P < .001; ****P < .0001. ns, not significant.

Article Snippet: Ten μg per 10 7 cells of antibody rabbit anti-EBF1 polyclonal IgG (ABE1294; Millipore, Burlington, MA) was hybridized to 70 μl Protein-G Dynabeads (Life Technologies) and ChIP-seq was performed as described in the supplemental Materials and methods.

Techniques: Expressing, In Vitro, Transduction, Plasmid Preparation, Standard Deviation, Transplantation Assay, Irradiation, Staining

Journal: Blood

Article Title: EBF1 and PAX5 control pro-B cell expansion via opposing regulation of the Myc gene

doi: 10.1182/blood.2020009564

Figure Lengend Snippet: The mouse Myc gene contains multiple EBF1-responsive enhancer elements. (A) UCSC Genome Browser view of the murine Myc locus and its distal interacting regions. The tracks display PAX5 (GSE126375) and EBF1 binding in 230-238 progenitor B cells, ATAC-accessibility (GSE92434), as well as a PLAC-sequencing derived virtual 4C tracks from Wt and Ebf1−/− FL-derived pro-B cells. Myc transcriptional start site ±2.5 kb was used as the viewpoint for the virtual 4C analysis. The previously defined BENC enhancer region29 is indicated by a dashed square. (B) Zoomed-in view of 3 specific regions in panel A with high PAX5 and EBF1 binding as well as ATAC accessibility in Wt pro-B cells and interaction with the Myc promoter. These regions were examined for the presence of histone modifications by reanalysis of ChIP- and CUT&RUN-sequencing data (GSE162858). The gray lines indicate the regions that are targeted for luciferase reporter activity assays. (C) Relative Firefly/Renilla (Prl0) luciferase activity obtained from reporter constructs in which the EBF1-binding regions described in panel B were cloned upstream of a basal Fos promoter in the absence (empty cDNA3) or presence of EBF1 in HeLa cells. Each dot represents one transfection, and the statistical analysis is based on a Student unpaired t test. **P < .01; ***P < .001; ****P < .0001.

Article Snippet: Ten μg per 10 7 cells of antibody rabbit anti-EBF1 polyclonal IgG (ABE1294; Millipore, Burlington, MA) was hybridized to 70 μl Protein-G Dynabeads (Life Technologies) and ChIP-seq was performed as described in the supplemental Materials and methods.

Techniques: Binding Assay, Sequencing, Derivative Assay, Luciferase, Activity Assay, Construct, Clone Assay, Transfection

Journal: Blood

Article Title: EBF1 and PAX5 control pro-B cell expansion via opposing regulation of the Myc gene

doi: 10.1182/blood.2020009564

Figure Lengend Snippet: EBF1 directly targets an essential binding site in the BENC enhancer region. (A) The sequence of known EBF1-binding sites and 6 predicted EBF1-binding sites within 5 putative Myc 3′enhancer elements as well as a potential EBF1-binding site in the 5′ region of Myc. The core binding site is indicated by a red box. (B) Autoradiogram displaying the result of an electrophoretic mobility shift assay experiment in which the binding of in vitro translated EBF1 to a radioactive labeled Cd79a promoter-EBF1 site is competed for by the addition of a 200-fold excess of nonlabeled putative EBF1-binding sites in Myc enhancers or the PAX5-binding site from the Cd19 promoter. The autoradiogram is representative of 2 independent experiments. (C) Schematic drawing of the targeting of CRISPR guides 106 and 107 to the EBF1-binding site in Myc E2. The DNA sequence of the EBF1 binding motif is depicted in yellow, and guides 106 (light brown) and 107 (dark brown) are shown pointing toward a 3′ NGG PAM sequence. The scale indicates the genomic location on mouse chromosome 15. (D) Myc quantitative reverse transcription PCR data from CD19+ iCas9 BM cells transduced with CRISPR guide 81 (control) or 107 (Myc E2) and subsequently treated with doxycycline (DOX) for 48 hours. Mean and standard deviation are shown; n = 6, from 3 individual samples from different mice. (E) Proliferation per 100 iCas9 CD19+ BM cells at 24, 48, and 96 hours after DOX administration in samples infected with gRNA constructs sg106 and 107 (targeting EBF1-binding site Myc E2), sg81 (targeting an EBF1 site linked to the Gfra2 gene), or sg112 (targeting the coding region of Myc). Mean and standard deviation are shown. *P < .05; **P < .01, Student t test compared with sg81), from 3 independent samples from different mice.

Article Snippet: Ten μg per 10 7 cells of antibody rabbit anti-EBF1 polyclonal IgG (ABE1294; Millipore, Burlington, MA) was hybridized to 70 μl Protein-G Dynabeads (Life Technologies) and ChIP-seq was performed as described in the supplemental Materials and methods.

Techniques: Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay, In Vitro, Labeling, CRISPR, Transduction, Standard Deviation, Infection, Construct

Journal: Blood

Article Title: EBF1 and PAX5 control pro-B cell expansion via opposing regulation of the Myc gene

doi: 10.1182/blood.2020009564

Figure Lengend Snippet: PAX5 acts as a negative regulator of cell proliferation and MYC function in pro-B cells. (A) Cell recovery 3 days after seeding of 2000 sorted GFP+CD45+ in vitro expanded FL cells from Ebf1−/− mice exposed to nuclear EBF1 by cultivation in 4-OHT for 4 or 7 days before sorting and reseeding in cultures in the absence of 4-OHT. Mean and standard deviation (SD) are shown; n = 5 to 6, from 2 independent experiments. Total cell recovery (B) and the fraction of CD19+ cells (C) recovered 4 days after seeding 2000 GFP+ Ebf1−/− FL cells transduced with either MIG-control, EBF1, or PAX5 encoding virus. Mean and SD are shown; n = 7, from 2 independent experiments. (D) Cell cycle data from Ebf1−/− FL cells transduced with MIG-control, EBF1, or PAX5 encoding virus as determined by FACS analysis. Mean and standard error of the mean are shown; n = 3. (E) Quantitative reverse transcription PCR analysis determining the levels of Myc transcripts in live sorted Wt FL cells or transduced EBF1-deficient cells as in panel B. Mean and SD are shown, n = 4, from 4 samples. (F) The ratio of MYC median fluorescent intensity (MFI) of each stage of the cell cycle compared with G0 in Ebf1-deficient cells transduced with EBF1- or PAX5-encoding vectors. Mean and standard error of the mean are shown; n = 3. (G) Gene set enrichment analysis of genes in HALLMARK_MYC_TARGET_V1 gene set based on RNA-sequencing data normalized per reads per kilobase of transcript, per million mapped reads from Ebf1−/− FL cells transduced with a control GFP or PAX5 encoding virus. (H) Representative histograms of Ebf1−/− BM cells rescued to the CD19+ pro–B-cell stage by transduction with either a conventional or ER-fused EBF1-encoding retrovirus and transduced with a Cas9-encoding virus alone or in combination with a Pax5-targeting gRNA. (I) MFI values for PAX5 levels as determined by flow cytometry. Mean and SD are shown; n = 4, from 4 individual samples. (J) Cell recovery after 3 days of in vitro culture of EBF1- or EBF1-ER–transduced cells expressing Cas9 alone or Cas9 in combination with gRNAs targeted to the Pax5 gene (gPAX5). Mean and SD are shown; n = 4, from 4 individual samples. For panels A-E and I-J, each dot indicates a data point, and the statistical analyses are based on a Student unpaired t test. *P < .05; **P < .01; ***P < .001; ****P < .0001. FDR = false discovery rate; NES = normalized enrichment score.

Article Snippet: Ten μg per 10 7 cells of antibody rabbit anti-EBF1 polyclonal IgG (ABE1294; Millipore, Burlington, MA) was hybridized to 70 μl Protein-G Dynabeads (Life Technologies) and ChIP-seq was performed as described in the supplemental Materials and methods.

Techniques: In Vitro, Standard Deviation, Transduction, RNA Sequencing Assay, Flow Cytometry, Expressing

Journal: Blood

Article Title: EBF1 and PAX5 control pro-B cell expansion via opposing regulation of the Myc gene

doi: 10.1182/blood.2020009564

Figure Lengend Snippet: Putative regulatory elements in the human MYC gene is targeted by EBF1 and PAX5 in pro-B ALL cells. (A) ChIP-sequencing, ATAC-sequencing, and PLAC-sequencing tracks for the human MYC (c-MYC) gene displayed in the WashU Epigenome Browser. Data were re-analyzed from Okuyama et al22 (GSE126300). (B) Schematic drawing of a model for regulatory loops controlling Myc expression in development.

Article Snippet: Ten μg per 10 7 cells of antibody rabbit anti-EBF1 polyclonal IgG (ABE1294; Millipore, Burlington, MA) was hybridized to 70 μl Protein-G Dynabeads (Life Technologies) and ChIP-seq was performed as described in the supplemental Materials and methods.

Techniques: ChIP-sequencing, Sequencing, Expressing