aebp2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc aebp2
    JAZF1-SUZ12 lacks interaction with JARID2, EPOP, and PALI1 due to loss of the SUZ12 N terminus (A) Models of FLAG-tagged (F) SUZ12, SUZ12Δ93, JAZF1-SUZ12, and JAZF1 constructs. (B) Immunoblots for FLAG, SUZ12 (N terminus), EZH2, JARID2, <t>AEBP2,</t> PCL2, EPOP, TRRAP, and β-actin in input (In.) and SUZ12 IPs from WT or Suz12 GT/GT ESCs stably expressing FLAG-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent IPs. (C) Immunoblots for FLAG, HA-PALI, and EZH2 in input and Strep-Tactin pull-downs (P) from NIH3T3 cells transfected with HA-PALI1 and FS2-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent pull-downs. See also <xref ref-type=Figure S1 . " width="250" height="auto" />
    Aebp2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "JAZF1-SUZ12 dysregulates PRC2 function and gene expression during cell differentiation"

    Article Title: JAZF1-SUZ12 dysregulates PRC2 function and gene expression during cell differentiation

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2022.110889

    JAZF1-SUZ12 lacks interaction with JARID2, EPOP, and PALI1 due to loss of the SUZ12 N terminus (A) Models of FLAG-tagged (F) SUZ12, SUZ12Δ93, JAZF1-SUZ12, and JAZF1 constructs. (B) Immunoblots for FLAG, SUZ12 (N terminus), EZH2, JARID2, AEBP2, PCL2, EPOP, TRRAP, and β-actin in input (In.) and SUZ12 IPs from WT or Suz12 GT/GT ESCs stably expressing FLAG-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent IPs. (C) Immunoblots for FLAG, HA-PALI, and EZH2 in input and Strep-Tactin pull-downs (P) from NIH3T3 cells transfected with HA-PALI1 and FS2-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent pull-downs. See also <xref ref-type=Figure S1 . " title="... Immunoblots for FLAG, SUZ12 (N terminus), EZH2, JARID2, AEBP2, PCL2, EPOP, TRRAP, and β-actin in input (In.) ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: JAZF1-SUZ12 lacks interaction with JARID2, EPOP, and PALI1 due to loss of the SUZ12 N terminus (A) Models of FLAG-tagged (F) SUZ12, SUZ12Δ93, JAZF1-SUZ12, and JAZF1 constructs. (B) Immunoblots for FLAG, SUZ12 (N terminus), EZH2, JARID2, AEBP2, PCL2, EPOP, TRRAP, and β-actin in input (In.) and SUZ12 IPs from WT or Suz12 GT/GT ESCs stably expressing FLAG-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent IPs. (C) Immunoblots for FLAG, HA-PALI, and EZH2 in input and Strep-Tactin pull-downs (P) from NIH3T3 cells transfected with HA-PALI1 and FS2-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent pull-downs. See also Figure S1 .

    Techniques Used: Construct, Western Blot, Stable Transfection, Expressing, Transfection


    Figure Legend Snippet:

    Techniques Used: Recombinant, Avidin-Biotin Assay, SYBR Green Assay, Sensitive Assay, Western Blot, Software

    aebp2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc aebp2
    JAZF1-SUZ12 lacks interaction with JARID2, EPOP, and PALI1 due to loss of the SUZ12 N terminus (A) Models of FLAG-tagged (F) SUZ12, SUZ12Δ93, JAZF1-SUZ12, and JAZF1 constructs. (B) Immunoblots for FLAG, SUZ12 (N terminus), EZH2, JARID2, <t>AEBP2,</t> PCL2, EPOP, TRRAP, and β-actin in input (In.) and SUZ12 IPs from WT or Suz12 GT/GT ESCs stably expressing FLAG-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent IPs. (C) Immunoblots for FLAG, HA-PALI, and EZH2 in input and Strep-Tactin pull-downs (P) from NIH3T3 cells transfected with HA-PALI1 and FS2-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent pull-downs. See also <xref ref-type=Figure S1 . " width="250" height="auto" />
    Aebp2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "JAZF1-SUZ12 dysregulates PRC2 function and gene expression during cell differentiation"

    Article Title: JAZF1-SUZ12 dysregulates PRC2 function and gene expression during cell differentiation

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2022.110889

    JAZF1-SUZ12 lacks interaction with JARID2, EPOP, and PALI1 due to loss of the SUZ12 N terminus (A) Models of FLAG-tagged (F) SUZ12, SUZ12Δ93, JAZF1-SUZ12, and JAZF1 constructs. (B) Immunoblots for FLAG, SUZ12 (N terminus), EZH2, JARID2, AEBP2, PCL2, EPOP, TRRAP, and β-actin in input (In.) and SUZ12 IPs from WT or Suz12 GT/GT ESCs stably expressing FLAG-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent IPs. (C) Immunoblots for FLAG, HA-PALI, and EZH2 in input and Strep-Tactin pull-downs (P) from NIH3T3 cells transfected with HA-PALI1 and FS2-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent pull-downs. See also <xref ref-type=Figure S1 . " title="... Immunoblots for FLAG, SUZ12 (N terminus), EZH2, JARID2, AEBP2, PCL2, EPOP, TRRAP, and β-actin in input (In.) ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: JAZF1-SUZ12 lacks interaction with JARID2, EPOP, and PALI1 due to loss of the SUZ12 N terminus (A) Models of FLAG-tagged (F) SUZ12, SUZ12Δ93, JAZF1-SUZ12, and JAZF1 constructs. (B) Immunoblots for FLAG, SUZ12 (N terminus), EZH2, JARID2, AEBP2, PCL2, EPOP, TRRAP, and β-actin in input (In.) and SUZ12 IPs from WT or Suz12 GT/GT ESCs stably expressing FLAG-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent IPs. (C) Immunoblots for FLAG, HA-PALI, and EZH2 in input and Strep-Tactin pull-downs (P) from NIH3T3 cells transfected with HA-PALI1 and FS2-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent pull-downs. See also Figure S1 .

    Techniques Used: Construct, Western Blot, Stable Transfection, Expressing, Transfection


    Figure Legend Snippet:

    Techniques Used: Recombinant, Avidin-Biotin Assay, SYBR Green Assay, Sensitive Assay, Western Blot, Software

    anti aebp2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti aebp2
    (A) Timeline of GapmeR-mediated gene knockdown and dox-induced Firre transgene expression. (B) Depiction of possible outcomes for Firre -responsive gene expression (red line) after knockdown of a regulatory factor (blue line) and induction of Firre transgene expression. (C) Relative gene expression of Firre and its targets Adgrg1 and Shf in Firre OE cells transfected with non-targeting control-(NTC), Ezh2 -, Suz12, Jarid2 -, <t>Aebp2</t> -, Wdr5 -, or G9a -targeting GapmeRs, and treated without (zero hours) or with (six hours) dox as determined by RT-qPCR. (D and E) smRNA FISH-based quantification of the distance between Shf intron and Firre transgene (D) and Shf intron and the five closest Firre exons (distance averaged) (E) in Firre -overexpression and Firre -rescue cells after 0, 2, 4, and 6 hours of dox treatment. The images below depict the rationale of the distance measurements.
    Anti Aebp2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The lncRNA Firre functions as a transcriptional activator from a distance"

    Article Title: The lncRNA Firre functions as a transcriptional activator from a distance

    Journal: bioRxiv

    doi: 10.1101/2022.05.15.492001

    (A) Timeline of GapmeR-mediated gene knockdown and dox-induced Firre transgene expression. (B) Depiction of possible outcomes for Firre -responsive gene expression (red line) after knockdown of a regulatory factor (blue line) and induction of Firre transgene expression. (C) Relative gene expression of Firre and its targets Adgrg1 and Shf in Firre OE cells transfected with non-targeting control-(NTC), Ezh2 -, Suz12, Jarid2 -, Aebp2 -, Wdr5 -, or G9a -targeting GapmeRs, and treated without (zero hours) or with (six hours) dox as determined by RT-qPCR. (D and E) smRNA FISH-based quantification of the distance between Shf intron and Firre transgene (D) and Shf intron and the five closest Firre exons (distance averaged) (E) in Firre -overexpression and Firre -rescue cells after 0, 2, 4, and 6 hours of dox treatment. The images below depict the rationale of the distance measurements.
    Figure Legend Snippet: (A) Timeline of GapmeR-mediated gene knockdown and dox-induced Firre transgene expression. (B) Depiction of possible outcomes for Firre -responsive gene expression (red line) after knockdown of a regulatory factor (blue line) and induction of Firre transgene expression. (C) Relative gene expression of Firre and its targets Adgrg1 and Shf in Firre OE cells transfected with non-targeting control-(NTC), Ezh2 -, Suz12, Jarid2 -, Aebp2 -, Wdr5 -, or G9a -targeting GapmeRs, and treated without (zero hours) or with (six hours) dox as determined by RT-qPCR. (D and E) smRNA FISH-based quantification of the distance between Shf intron and Firre transgene (D) and Shf intron and the five closest Firre exons (distance averaged) (E) in Firre -overexpression and Firre -rescue cells after 0, 2, 4, and 6 hours of dox treatment. The images below depict the rationale of the distance measurements.

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Over Expression

    e2f2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc e2f2
    PCR primer sequences
    E2f2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "MicroRNA-631 deriving from bone marrow mesenchymal stem cell exosomes facilitates the malignant behavior of non-small cell lung cancer via modulating the E2F family of transcription factor 2/phosphatidylinositol 3‐kinase/Akt signaling pathway"

    Article Title: MicroRNA-631 deriving from bone marrow mesenchymal stem cell exosomes facilitates the malignant behavior of non-small cell lung cancer via modulating the E2F family of transcription factor 2/phosphatidylinositol 3‐kinase/Akt signaling pathway

    Journal: Bioengineered

    doi: 10.1080/21655979.2022.2036891

    PCR primer sequences
    Figure Legend Snippet: PCR primer sequences

    Techniques Used:

    E2F2 is the target gene of miR-631, and elevated E2F2 facilitates the malignant behavior of NSCLC.
    Figure Legend Snippet: E2F2 is the target gene of miR-631, and elevated E2F2 facilitates the malignant behavior of NSCLC.

    Techniques Used:

    MiR-631 exerts an influence on the malignant behavior of NSCLC via modulating E2F2.
    Figure Legend Snippet: MiR-631 exerts an influence on the malignant behavior of NSCLC via modulating E2F2.

    Techniques Used:

    BMSCs-Exo exerts an influence on the malignant behavior of NSCLC via the miR-631/E2F2 axis.
    Figure Legend Snippet: BMSCs-Exo exerts an influence on the malignant behavior of NSCLC via the miR-631/E2F2 axis.

    Techniques Used:

    s rrid ab 2798398  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc s rrid ab 2798398
    S Rrid Ab 2798398, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti aebp2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti aebp2
    ( A ) Nuclear <t>Aebp2</t> detection by immunofluorescence in postzygotic genome activation (postZGA) zebrafish embryos (4.3 hr post fertilization [hpf]). No Aebp2 staining was detected at preZGA (2.5 hpf) or ZGA (3.5 hpf). Bottom row: The dashed square indicates the field of view in upper panels. One of three biological replicates is shown. ( B ) Aebp2 binding at promoters during postZGA overlaps and scales with occupancy of Rnf2, H2Aub1, and H3K27me3. Promoter clusters from were utilized to plot heatmaps. ( C ) Genome browser screenshots of chromatin immunoprecipitation (ChIP)-seq at representative promoter loci from clusters in ( B ). ( D ) Aebp2 binding at enhancers during postZGA overlaps with occupancy of Rnf2, H2Aub1, and H3K27me3. Enhancer clusters from were utilized to plot heatmaps. ( E ) Genome browser screenshots of ChIP-seq enrichment at representative enhancer loci from clusters in ( D ).
    Anti Aebp2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Establishment of developmental gene silencing by ordered polycomb complex recruitment in early zebrafish embryos"

    Article Title: Establishment of developmental gene silencing by ordered polycomb complex recruitment in early zebrafish embryos

    Journal: eLife

    doi: 10.7554/eLife.67738

    ( A ) Nuclear Aebp2 detection by immunofluorescence in postzygotic genome activation (postZGA) zebrafish embryos (4.3 hr post fertilization [hpf]). No Aebp2 staining was detected at preZGA (2.5 hpf) or ZGA (3.5 hpf). Bottom row: The dashed square indicates the field of view in upper panels. One of three biological replicates is shown. ( B ) Aebp2 binding at promoters during postZGA overlaps and scales with occupancy of Rnf2, H2Aub1, and H3K27me3. Promoter clusters from were utilized to plot heatmaps. ( C ) Genome browser screenshots of chromatin immunoprecipitation (ChIP)-seq at representative promoter loci from clusters in ( B ). ( D ) Aebp2 binding at enhancers during postZGA overlaps with occupancy of Rnf2, H2Aub1, and H3K27me3. Enhancer clusters from were utilized to plot heatmaps. ( E ) Genome browser screenshots of ChIP-seq enrichment at representative enhancer loci from clusters in ( D ).
    Figure Legend Snippet: ( A ) Nuclear Aebp2 detection by immunofluorescence in postzygotic genome activation (postZGA) zebrafish embryos (4.3 hr post fertilization [hpf]). No Aebp2 staining was detected at preZGA (2.5 hpf) or ZGA (3.5 hpf). Bottom row: The dashed square indicates the field of view in upper panels. One of three biological replicates is shown. ( B ) Aebp2 binding at promoters during postZGA overlaps and scales with occupancy of Rnf2, H2Aub1, and H3K27me3. Promoter clusters from were utilized to plot heatmaps. ( C ) Genome browser screenshots of chromatin immunoprecipitation (ChIP)-seq at representative promoter loci from clusters in ( B ). ( D ) Aebp2 binding at enhancers during postZGA overlaps with occupancy of Rnf2, H2Aub1, and H3K27me3. Enhancer clusters from were utilized to plot heatmaps. ( E ) Genome browser screenshots of ChIP-seq enrichment at representative enhancer loci from clusters in ( D ).

    Techniques Used: Immunofluorescence, Activation Assay, Staining, Binding Assay, Chromatin Immunoprecipitation, ChIP-sequencing

    ( A ) Abundance of aebp2 mRNA in early zebrafish embryos measured by RNA-sequencing. Increased Aebp2 protein levels from preZGA to postZGA observed in are not explained by increased aebp2 mRNA levels (black bars). Levels of rnf2 mRNA are plotted for comparison (gray bars). RNA-seq data was collected from http://www.ebiac.uk/gxa/experiments/E-ERAD-475 . ( B ) Aebp2 ChIP-qPCR demonstrates enrichment of Aebp2 at developmental gene promoters (pax6a, vsx1, isl1) in postZGA stage embryos (4.3 hr post fertilization [hpf]). Housekeeping gene promoters (idh3g, pcf11, lman2) were used as negative control regions. Note: Promoter regions assayed here are either robustly marked by H2Aub1 (pax6a, vsx1, isl1) or lack H2Aub1 (idh3g, pcf11, lman2). Three biological replicates were conducted for ChIP. For qPCR, three technical replicates were conducted on each ChIP biological replicate. ( C ) Genome-wide correlation matrix of Aebp2 ChIP-seq replicates and input from postZGA (4.3 hpf) zebrafish embryos. Heatmap matrix displays Pearson correlation coefficients. ( D ) Genome browser screenshots of representative promoters with ChIP-seq enrichment demonstrating binding of Aebp2 at promoters bearing Rnf2, H2Aub1, and H3K27me3 at postZGA (4.3 hpf). ( E ) Genome browser screenshots of representative enhancers with ChIP-seq enrichment demonstrating binding of Aebp2 at enhancers bearing Rnf2, H2Aub1, and H3K27me3 at postZGA (4.3 hpf).
    Figure Legend Snippet: ( A ) Abundance of aebp2 mRNA in early zebrafish embryos measured by RNA-sequencing. Increased Aebp2 protein levels from preZGA to postZGA observed in are not explained by increased aebp2 mRNA levels (black bars). Levels of rnf2 mRNA are plotted for comparison (gray bars). RNA-seq data was collected from http://www.ebiac.uk/gxa/experiments/E-ERAD-475 . ( B ) Aebp2 ChIP-qPCR demonstrates enrichment of Aebp2 at developmental gene promoters (pax6a, vsx1, isl1) in postZGA stage embryos (4.3 hr post fertilization [hpf]). Housekeeping gene promoters (idh3g, pcf11, lman2) were used as negative control regions. Note: Promoter regions assayed here are either robustly marked by H2Aub1 (pax6a, vsx1, isl1) or lack H2Aub1 (idh3g, pcf11, lman2). Three biological replicates were conducted for ChIP. For qPCR, three technical replicates were conducted on each ChIP biological replicate. ( C ) Genome-wide correlation matrix of Aebp2 ChIP-seq replicates and input from postZGA (4.3 hpf) zebrafish embryos. Heatmap matrix displays Pearson correlation coefficients. ( D ) Genome browser screenshots of representative promoters with ChIP-seq enrichment demonstrating binding of Aebp2 at promoters bearing Rnf2, H2Aub1, and H3K27me3 at postZGA (4.3 hpf). ( E ) Genome browser screenshots of representative enhancers with ChIP-seq enrichment demonstrating binding of Aebp2 at enhancers bearing Rnf2, H2Aub1, and H3K27me3 at postZGA (4.3 hpf).

    Techniques Used: RNA Sequencing Assay, Negative Control, Genome Wide, ChIP-sequencing, Binding Assay

    ( A ) Genome-wide correlation matrix of Jarid2 ChIP-seq replicates and input from postZGA (4.3 hr post fertilization [hpf]) zebrafish embryos. Heatmap matrix displays Pearson correlation coefficients. ( B ) Venn diagram of gene promoters with peaks of Aebp2 or Jarid2. ( C ) Top three enriched GO-term categories from the genes depicted in ( B ). ( D ) Genome browser screenshots of representative promoters with ChIP-seq enrichment of H2Aub1, Aebp2, Jarid2, and H3K27me3 at postZGA (4.3 hpf). ( E ) Venn diagram of enhancer loci with peaks of Aebp2 or Jarid2. ( F ) Genome browser screenshots of representative enhancers with ChIP-seq enrichment of H2Aub1, Aebp2, Jarid2, and H3K27me3 at postZGA (4.3 hpf).
    Figure Legend Snippet: ( A ) Genome-wide correlation matrix of Jarid2 ChIP-seq replicates and input from postZGA (4.3 hr post fertilization [hpf]) zebrafish embryos. Heatmap matrix displays Pearson correlation coefficients. ( B ) Venn diagram of gene promoters with peaks of Aebp2 or Jarid2. ( C ) Top three enriched GO-term categories from the genes depicted in ( B ). ( D ) Genome browser screenshots of representative promoters with ChIP-seq enrichment of H2Aub1, Aebp2, Jarid2, and H3K27me3 at postZGA (4.3 hpf). ( E ) Venn diagram of enhancer loci with peaks of Aebp2 or Jarid2. ( F ) Genome browser screenshots of representative enhancers with ChIP-seq enrichment of H2Aub1, Aebp2, Jarid2, and H3K27me3 at postZGA (4.3 hpf).

    Techniques Used: Genome Wide, ChIP-sequencing

    ( A ) Experimental design of drug treatments to inhibit Rnf2 activity. Embryos at the one-cell stage were added to media containing either PRT4165 (150 μM) or DMSO and raised until 4 hr post fertilization (hpf). ( B ) PRT4165 treatment of embryos confers bulk loss of H2Aub1 at 4 hpf. Left: Western blot for H2Aub1 in 4 hpf embryos treated with DMSO (vehicle) or 150 μM PRT4165 (Rnf2 inhibitor). Right: Quantification western blot in left panel. ( C ) Impact of Rnf2 inhibition on Aebp2 genomic localization and H3K27me3. K-means clustering of Aebp2 and H3K27me3 chromatin immunoprecipitation (ChIP)-seq enrichment at all loci with called peaks in any of the datasets plotted. Embryos were treated from the one-cell stage with either DMSO or 150 μM PRT4165 and harvested at 4 hpf for ChIP analysis. H3K27me3 ChIP-seq from untreated embryos at 4.3 hpf is plotted as a comparitor. ( D ) Impact of Rnf2 inhibition on gene expression. Volcano plot of RNA-seq data from PRT4165-treated vs. untreated embryos (4 hpf). Green and red data points signify transcripts with p-values < 0.01 and at least a 3-fold change (increase or decrease) in expression, respectively. Marquee upregulated genes encoding developmental transcription factors are labelled. ( E ) Genome browser screenshots of representative developmental genes which, upon Rnf2 inhibition, lose Aebp2 binding and H3K27me3 marking, and become transcriptionally active. Figure 4—source data 1. Uncropped western blots for panel B.
    Figure Legend Snippet: ( A ) Experimental design of drug treatments to inhibit Rnf2 activity. Embryos at the one-cell stage were added to media containing either PRT4165 (150 μM) or DMSO and raised until 4 hr post fertilization (hpf). ( B ) PRT4165 treatment of embryos confers bulk loss of H2Aub1 at 4 hpf. Left: Western blot for H2Aub1 in 4 hpf embryos treated with DMSO (vehicle) or 150 μM PRT4165 (Rnf2 inhibitor). Right: Quantification western blot in left panel. ( C ) Impact of Rnf2 inhibition on Aebp2 genomic localization and H3K27me3. K-means clustering of Aebp2 and H3K27me3 chromatin immunoprecipitation (ChIP)-seq enrichment at all loci with called peaks in any of the datasets plotted. Embryos were treated from the one-cell stage with either DMSO or 150 μM PRT4165 and harvested at 4 hpf for ChIP analysis. H3K27me3 ChIP-seq from untreated embryos at 4.3 hpf is plotted as a comparitor. ( D ) Impact of Rnf2 inhibition on gene expression. Volcano plot of RNA-seq data from PRT4165-treated vs. untreated embryos (4 hpf). Green and red data points signify transcripts with p-values < 0.01 and at least a 3-fold change (increase or decrease) in expression, respectively. Marquee upregulated genes encoding developmental transcription factors are labelled. ( E ) Genome browser screenshots of representative developmental genes which, upon Rnf2 inhibition, lose Aebp2 binding and H3K27me3 marking, and become transcriptionally active. Figure 4—source data 1. Uncropped western blots for panel B.

    Techniques Used: Activity Assay, Western Blot, Inhibition, Chromatin Immunoprecipitation, ChIP-sequencing, Expressing, RNA Sequencing Assay, Binding Assay

    ( A ) Treatment of zebrafish embryos with PRT4165 (150 μM) beginning at the one-cell stage results in developmental arrest at 4 hr post fertilization (hpf). Removal of PRT4165 at 4 hpf failed to restore normal developmental progression to embryos (lower right, ‘wash out’). ( B ) Aebp2 ChIP-qPCR in postzygotic genome activation (postZGA) (4 hpf) embryos treated with DMSO (black bars) or PRT4165 (150 μM, red bars). Binding of Aebp2 to developmental gene promoters (pax6a, vsx1, isl1) is lost upon PRC1 inhibition. ( C ) Genome-wide correlation matrices of Aebp2 ChIP-seq replicates and input from postZGA (4 hpf) zebrafish embryos treated with DMSO (left) or PRT4165 (150 μM, right). Heatmap matrices display Pearson correlation coefficients. ( D ) Genome-wide correlation matrices of H3K27me3 ChIP-seq replicates and input from postZGA (4 hpf) zebrafish embryos treated with DMSO or PRT4165 (150 μM, right). Heatmap matrices display Pearson correlation coefficients. ( E ) Genome-wide correlation matrix of RNA-seq replicates from postZGA (4 hpf) zebrafish embryos treated with DMSO or PRT4165. Heatmap matrix displays Pearson correlation coefficients. ( F ) GO-terms associated with upregulated and downregulated transcripts depicted in . GO-term analysis was performed with DAVID .
    Figure Legend Snippet: ( A ) Treatment of zebrafish embryos with PRT4165 (150 μM) beginning at the one-cell stage results in developmental arrest at 4 hr post fertilization (hpf). Removal of PRT4165 at 4 hpf failed to restore normal developmental progression to embryos (lower right, ‘wash out’). ( B ) Aebp2 ChIP-qPCR in postzygotic genome activation (postZGA) (4 hpf) embryos treated with DMSO (black bars) or PRT4165 (150 μM, red bars). Binding of Aebp2 to developmental gene promoters (pax6a, vsx1, isl1) is lost upon PRC1 inhibition. ( C ) Genome-wide correlation matrices of Aebp2 ChIP-seq replicates and input from postZGA (4 hpf) zebrafish embryos treated with DMSO (left) or PRT4165 (150 μM, right). Heatmap matrices display Pearson correlation coefficients. ( D ) Genome-wide correlation matrices of H3K27me3 ChIP-seq replicates and input from postZGA (4 hpf) zebrafish embryos treated with DMSO or PRT4165 (150 μM, right). Heatmap matrices display Pearson correlation coefficients. ( E ) Genome-wide correlation matrix of RNA-seq replicates from postZGA (4 hpf) zebrafish embryos treated with DMSO or PRT4165. Heatmap matrix displays Pearson correlation coefficients. ( F ) GO-terms associated with upregulated and downregulated transcripts depicted in . GO-term analysis was performed with DAVID .

    Techniques Used: Activation Assay, Binding Assay, Inhibition, Genome Wide, ChIP-sequencing, RNA Sequencing Assay

    ( A ) Genome browser screenshots of representative de-repressed genes upon PRC1 inhibition. Both six3b and six2b are bound by Rnf2 and marked by H2Aub1 from prezygotic genome activation (preZGA) onward. At postZGA these genes normally attract binding by Aebp2 and acquire H3K27me3. Inhibition of Rnf2 (via PRT4165) causes failed Aebp2 binding, and prevention of H3K27me3 establishment. Notably, six3b is precociously expressed upon loss of H2Aub1. ( B ) Genome browser screenshots of representative loci that gain ectopic Aebp2 binding (left) or H3K27me3 marking (right) upon PRC1 inhibition. Ectopic binding events for Aebp2 or H3K27me3 appear to be mutually exclusive. Dotted boxes denote region of ectopic binding or marking by Aebp2 or H3K27me3, respectively. ( C ) Approximately 16.6% of genes normally silenced by H2Aub1 promoter marking are transcriptionally upregulated upon inhibition of Rnf2. Venn diagram displays the overlap between the gene promoters normally marked by H2Aub1 and protein coding genes that are transcriptionally upregulated (p-value < 0.001, >1.5-fold change) after Rnf2 inhibition. Here, H2Aub1 marked promoters are defined as regions ±2 Kb from transcription start sites and having peak(s) called by MACS2 (q-value cutoff 0.01) of H2Aub1 from 4.3 hpf H2Aub1 ChIP-seq.
    Figure Legend Snippet: ( A ) Genome browser screenshots of representative de-repressed genes upon PRC1 inhibition. Both six3b and six2b are bound by Rnf2 and marked by H2Aub1 from prezygotic genome activation (preZGA) onward. At postZGA these genes normally attract binding by Aebp2 and acquire H3K27me3. Inhibition of Rnf2 (via PRT4165) causes failed Aebp2 binding, and prevention of H3K27me3 establishment. Notably, six3b is precociously expressed upon loss of H2Aub1. ( B ) Genome browser screenshots of representative loci that gain ectopic Aebp2 binding (left) or H3K27me3 marking (right) upon PRC1 inhibition. Ectopic binding events for Aebp2 or H3K27me3 appear to be mutually exclusive. Dotted boxes denote region of ectopic binding or marking by Aebp2 or H3K27me3, respectively. ( C ) Approximately 16.6% of genes normally silenced by H2Aub1 promoter marking are transcriptionally upregulated upon inhibition of Rnf2. Venn diagram displays the overlap between the gene promoters normally marked by H2Aub1 and protein coding genes that are transcriptionally upregulated (p-value < 0.001, >1.5-fold change) after Rnf2 inhibition. Here, H2Aub1 marked promoters are defined as regions ±2 Kb from transcription start sites and having peak(s) called by MACS2 (q-value cutoff 0.01) of H2Aub1 from 4.3 hpf H2Aub1 ChIP-seq.

    Techniques Used: Inhibition, Activation Assay, Binding Assay, ChIP-sequencing

    Prior to zygotic genome activation (ZGA), Rnf2-PRC1 is recruited by transcription factors (TFs) to promoters (shown) and enhancers (not shown) of developmental genes bearing Placeholder nucleosomes (H2A.Z(FV), H3K4me1, H3K27ac). Rnf2-PRC1 deposition of H2Aub1 recruits Aebp2-PRC2 to catalyze H3K27me3 addition. H2Aub1 ablation (via Rnf2 inhibition) eliminates Aebp2-PRC2 recruitment and prevents H3K27me3 establishment. Notably, H2Aub1 loss causes precocious transcription of certain developmental genes after ZGA, identifying H2Aub1 as a critical component of silencing at ZGA.
    Figure Legend Snippet: Prior to zygotic genome activation (ZGA), Rnf2-PRC1 is recruited by transcription factors (TFs) to promoters (shown) and enhancers (not shown) of developmental genes bearing Placeholder nucleosomes (H2A.Z(FV), H3K4me1, H3K27ac). Rnf2-PRC1 deposition of H2Aub1 recruits Aebp2-PRC2 to catalyze H3K27me3 addition. H2Aub1 ablation (via Rnf2 inhibition) eliminates Aebp2-PRC2 recruitment and prevents H3K27me3 establishment. Notably, H2Aub1 loss causes precocious transcription of certain developmental genes after ZGA, identifying H2Aub1 as a critical component of silencing at ZGA.

    Techniques Used: Activation Assay, Inhibition


    Figure Legend Snippet:

    Techniques Used: Software

    anti aebp2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti aebp2
    ( A ) Nuclear <t>Aebp2</t> detection by immunofluorescence in postzygotic genome activation (postZGA) zebrafish embryos (4.3 hr post fertilization [hpf]). No Aebp2 staining was detected at preZGA (2.5 hpf) or ZGA (3.5 hpf). Bottom row: The dashed square indicates the field of view in upper panels. One of three biological replicates is shown. ( B ) Aebp2 binding at promoters during postZGA overlaps and scales with occupancy of Rnf2, H2Aub1, and H3K27me3. Promoter clusters from were utilized to plot heatmaps. ( C ) Genome browser screenshots of chromatin immunoprecipitation (ChIP)-seq at representative promoter loci from clusters in ( B ). ( D ) Aebp2 binding at enhancers during postZGA overlaps with occupancy of Rnf2, H2Aub1, and H3K27me3. Enhancer clusters from were utilized to plot heatmaps. ( E ) Genome browser screenshots of ChIP-seq enrichment at representative enhancer loci from clusters in ( D ).
    Anti Aebp2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Establishment of developmental gene silencing by ordered polycomb complex recruitment in early zebrafish embryos"

    Article Title: Establishment of developmental gene silencing by ordered polycomb complex recruitment in early zebrafish embryos

    Journal: eLife

    doi: 10.7554/eLife.67738

    ( A ) Nuclear Aebp2 detection by immunofluorescence in postzygotic genome activation (postZGA) zebrafish embryos (4.3 hr post fertilization [hpf]). No Aebp2 staining was detected at preZGA (2.5 hpf) or ZGA (3.5 hpf). Bottom row: The dashed square indicates the field of view in upper panels. One of three biological replicates is shown. ( B ) Aebp2 binding at promoters during postZGA overlaps and scales with occupancy of Rnf2, H2Aub1, and H3K27me3. Promoter clusters from were utilized to plot heatmaps. ( C ) Genome browser screenshots of chromatin immunoprecipitation (ChIP)-seq at representative promoter loci from clusters in ( B ). ( D ) Aebp2 binding at enhancers during postZGA overlaps with occupancy of Rnf2, H2Aub1, and H3K27me3. Enhancer clusters from were utilized to plot heatmaps. ( E ) Genome browser screenshots of ChIP-seq enrichment at representative enhancer loci from clusters in ( D ).
    Figure Legend Snippet: ( A ) Nuclear Aebp2 detection by immunofluorescence in postzygotic genome activation (postZGA) zebrafish embryos (4.3 hr post fertilization [hpf]). No Aebp2 staining was detected at preZGA (2.5 hpf) or ZGA (3.5 hpf). Bottom row: The dashed square indicates the field of view in upper panels. One of three biological replicates is shown. ( B ) Aebp2 binding at promoters during postZGA overlaps and scales with occupancy of Rnf2, H2Aub1, and H3K27me3. Promoter clusters from were utilized to plot heatmaps. ( C ) Genome browser screenshots of chromatin immunoprecipitation (ChIP)-seq at representative promoter loci from clusters in ( B ). ( D ) Aebp2 binding at enhancers during postZGA overlaps with occupancy of Rnf2, H2Aub1, and H3K27me3. Enhancer clusters from were utilized to plot heatmaps. ( E ) Genome browser screenshots of ChIP-seq enrichment at representative enhancer loci from clusters in ( D ).

    Techniques Used: Immunofluorescence, Activation Assay, Staining, Binding Assay, Chromatin Immunoprecipitation, ChIP-sequencing

    ( A ) Abundance of aebp2 mRNA in early zebrafish embryos measured by RNA-sequencing. Increased Aebp2 protein levels from preZGA to postZGA observed in are not explained by increased aebp2 mRNA levels (black bars). Levels of rnf2 mRNA are plotted for comparison (gray bars). RNA-seq data was collected from http://www.ebiac.uk/gxa/experiments/E-ERAD-475 . ( B ) Aebp2 ChIP-qPCR demonstrates enrichment of Aebp2 at developmental gene promoters (pax6a, vsx1, isl1) in postZGA stage embryos (4.3 hr post fertilization [hpf]). Housekeeping gene promoters (idh3g, pcf11, lman2) were used as negative control regions. Note: Promoter regions assayed here are either robustly marked by H2Aub1 (pax6a, vsx1, isl1) or lack H2Aub1 (idh3g, pcf11, lman2). Three biological replicates were conducted for ChIP. For qPCR, three technical replicates were conducted on each ChIP biological replicate. ( C ) Genome-wide correlation matrix of Aebp2 ChIP-seq replicates and input from postZGA (4.3 hpf) zebrafish embryos. Heatmap matrix displays Pearson correlation coefficients. ( D ) Genome browser screenshots of representative promoters with ChIP-seq enrichment demonstrating binding of Aebp2 at promoters bearing Rnf2, H2Aub1, and H3K27me3 at postZGA (4.3 hpf). ( E ) Genome browser screenshots of representative enhancers with ChIP-seq enrichment demonstrating binding of Aebp2 at enhancers bearing Rnf2, H2Aub1, and H3K27me3 at postZGA (4.3 hpf).
    Figure Legend Snippet: ( A ) Abundance of aebp2 mRNA in early zebrafish embryos measured by RNA-sequencing. Increased Aebp2 protein levels from preZGA to postZGA observed in are not explained by increased aebp2 mRNA levels (black bars). Levels of rnf2 mRNA are plotted for comparison (gray bars). RNA-seq data was collected from http://www.ebiac.uk/gxa/experiments/E-ERAD-475 . ( B ) Aebp2 ChIP-qPCR demonstrates enrichment of Aebp2 at developmental gene promoters (pax6a, vsx1, isl1) in postZGA stage embryos (4.3 hr post fertilization [hpf]). Housekeeping gene promoters (idh3g, pcf11, lman2) were used as negative control regions. Note: Promoter regions assayed here are either robustly marked by H2Aub1 (pax6a, vsx1, isl1) or lack H2Aub1 (idh3g, pcf11, lman2). Three biological replicates were conducted for ChIP. For qPCR, three technical replicates were conducted on each ChIP biological replicate. ( C ) Genome-wide correlation matrix of Aebp2 ChIP-seq replicates and input from postZGA (4.3 hpf) zebrafish embryos. Heatmap matrix displays Pearson correlation coefficients. ( D ) Genome browser screenshots of representative promoters with ChIP-seq enrichment demonstrating binding of Aebp2 at promoters bearing Rnf2, H2Aub1, and H3K27me3 at postZGA (4.3 hpf). ( E ) Genome browser screenshots of representative enhancers with ChIP-seq enrichment demonstrating binding of Aebp2 at enhancers bearing Rnf2, H2Aub1, and H3K27me3 at postZGA (4.3 hpf).

    Techniques Used: RNA Sequencing Assay, Negative Control, Genome Wide, ChIP-sequencing, Binding Assay

    ( A ) Genome-wide correlation matrix of Jarid2 ChIP-seq replicates and input from postZGA (4.3 hr post fertilization [hpf]) zebrafish embryos. Heatmap matrix displays Pearson correlation coefficients. ( B ) Venn diagram of gene promoters with peaks of Aebp2 or Jarid2. ( C ) Top three enriched GO-term categories from the genes depicted in ( B ). ( D ) Genome browser screenshots of representative promoters with ChIP-seq enrichment of H2Aub1, Aebp2, Jarid2, and H3K27me3 at postZGA (4.3 hpf). ( E ) Venn diagram of enhancer loci with peaks of Aebp2 or Jarid2. ( F ) Genome browser screenshots of representative enhancers with ChIP-seq enrichment of H2Aub1, Aebp2, Jarid2, and H3K27me3 at postZGA (4.3 hpf).
    Figure Legend Snippet: ( A ) Genome-wide correlation matrix of Jarid2 ChIP-seq replicates and input from postZGA (4.3 hr post fertilization [hpf]) zebrafish embryos. Heatmap matrix displays Pearson correlation coefficients. ( B ) Venn diagram of gene promoters with peaks of Aebp2 or Jarid2. ( C ) Top three enriched GO-term categories from the genes depicted in ( B ). ( D ) Genome browser screenshots of representative promoters with ChIP-seq enrichment of H2Aub1, Aebp2, Jarid2, and H3K27me3 at postZGA (4.3 hpf). ( E ) Venn diagram of enhancer loci with peaks of Aebp2 or Jarid2. ( F ) Genome browser screenshots of representative enhancers with ChIP-seq enrichment of H2Aub1, Aebp2, Jarid2, and H3K27me3 at postZGA (4.3 hpf).

    Techniques Used: Genome Wide, ChIP-sequencing

    ( A ) Experimental design of drug treatments to inhibit Rnf2 activity. Embryos at the one-cell stage were added to media containing either PRT4165 (150 μM) or DMSO and raised until 4 hr post fertilization (hpf). ( B ) PRT4165 treatment of embryos confers bulk loss of H2Aub1 at 4 hpf. Left: Western blot for H2Aub1 in 4 hpf embryos treated with DMSO (vehicle) or 150 μM PRT4165 (Rnf2 inhibitor). Right: Quantification western blot in left panel. ( C ) Impact of Rnf2 inhibition on Aebp2 genomic localization and H3K27me3. K-means clustering of Aebp2 and H3K27me3 chromatin immunoprecipitation (ChIP)-seq enrichment at all loci with called peaks in any of the datasets plotted. Embryos were treated from the one-cell stage with either DMSO or 150 μM PRT4165 and harvested at 4 hpf for ChIP analysis. H3K27me3 ChIP-seq from untreated embryos at 4.3 hpf is plotted as a comparitor. ( D ) Impact of Rnf2 inhibition on gene expression. Volcano plot of RNA-seq data from PRT4165-treated vs. untreated embryos (4 hpf). Green and red data points signify transcripts with p-values < 0.01 and at least a 3-fold change (increase or decrease) in expression, respectively. Marquee upregulated genes encoding developmental transcription factors are labelled. ( E ) Genome browser screenshots of representative developmental genes which, upon Rnf2 inhibition, lose Aebp2 binding and H3K27me3 marking, and become transcriptionally active. Figure 4—source data 1. Uncropped western blots for panel B.
    Figure Legend Snippet: ( A ) Experimental design of drug treatments to inhibit Rnf2 activity. Embryos at the one-cell stage were added to media containing either PRT4165 (150 μM) or DMSO and raised until 4 hr post fertilization (hpf). ( B ) PRT4165 treatment of embryos confers bulk loss of H2Aub1 at 4 hpf. Left: Western blot for H2Aub1 in 4 hpf embryos treated with DMSO (vehicle) or 150 μM PRT4165 (Rnf2 inhibitor). Right: Quantification western blot in left panel. ( C ) Impact of Rnf2 inhibition on Aebp2 genomic localization and H3K27me3. K-means clustering of Aebp2 and H3K27me3 chromatin immunoprecipitation (ChIP)-seq enrichment at all loci with called peaks in any of the datasets plotted. Embryos were treated from the one-cell stage with either DMSO or 150 μM PRT4165 and harvested at 4 hpf for ChIP analysis. H3K27me3 ChIP-seq from untreated embryos at 4.3 hpf is plotted as a comparitor. ( D ) Impact of Rnf2 inhibition on gene expression. Volcano plot of RNA-seq data from PRT4165-treated vs. untreated embryos (4 hpf). Green and red data points signify transcripts with p-values < 0.01 and at least a 3-fold change (increase or decrease) in expression, respectively. Marquee upregulated genes encoding developmental transcription factors are labelled. ( E ) Genome browser screenshots of representative developmental genes which, upon Rnf2 inhibition, lose Aebp2 binding and H3K27me3 marking, and become transcriptionally active. Figure 4—source data 1. Uncropped western blots for panel B.

    Techniques Used: Activity Assay, Western Blot, Inhibition, Chromatin Immunoprecipitation, ChIP-sequencing, Expressing, RNA Sequencing Assay, Binding Assay

    ( A ) Treatment of zebrafish embryos with PRT4165 (150 μM) beginning at the one-cell stage results in developmental arrest at 4 hr post fertilization (hpf). Removal of PRT4165 at 4 hpf failed to restore normal developmental progression to embryos (lower right, ‘wash out’). ( B ) Aebp2 ChIP-qPCR in postzygotic genome activation (postZGA) (4 hpf) embryos treated with DMSO (black bars) or PRT4165 (150 μM, red bars). Binding of Aebp2 to developmental gene promoters (pax6a, vsx1, isl1) is lost upon PRC1 inhibition. ( C ) Genome-wide correlation matrices of Aebp2 ChIP-seq replicates and input from postZGA (4 hpf) zebrafish embryos treated with DMSO (left) or PRT4165 (150 μM, right). Heatmap matrices display Pearson correlation coefficients. ( D ) Genome-wide correlation matrices of H3K27me3 ChIP-seq replicates and input from postZGA (4 hpf) zebrafish embryos treated with DMSO or PRT4165 (150 μM, right). Heatmap matrices display Pearson correlation coefficients. ( E ) Genome-wide correlation matrix of RNA-seq replicates from postZGA (4 hpf) zebrafish embryos treated with DMSO or PRT4165. Heatmap matrix displays Pearson correlation coefficients. ( F ) GO-terms associated with upregulated and downregulated transcripts depicted in . GO-term analysis was performed with DAVID .
    Figure Legend Snippet: ( A ) Treatment of zebrafish embryos with PRT4165 (150 μM) beginning at the one-cell stage results in developmental arrest at 4 hr post fertilization (hpf). Removal of PRT4165 at 4 hpf failed to restore normal developmental progression to embryos (lower right, ‘wash out’). ( B ) Aebp2 ChIP-qPCR in postzygotic genome activation (postZGA) (4 hpf) embryos treated with DMSO (black bars) or PRT4165 (150 μM, red bars). Binding of Aebp2 to developmental gene promoters (pax6a, vsx1, isl1) is lost upon PRC1 inhibition. ( C ) Genome-wide correlation matrices of Aebp2 ChIP-seq replicates and input from postZGA (4 hpf) zebrafish embryos treated with DMSO (left) or PRT4165 (150 μM, right). Heatmap matrices display Pearson correlation coefficients. ( D ) Genome-wide correlation matrices of H3K27me3 ChIP-seq replicates and input from postZGA (4 hpf) zebrafish embryos treated with DMSO or PRT4165 (150 μM, right). Heatmap matrices display Pearson correlation coefficients. ( E ) Genome-wide correlation matrix of RNA-seq replicates from postZGA (4 hpf) zebrafish embryos treated with DMSO or PRT4165. Heatmap matrix displays Pearson correlation coefficients. ( F ) GO-terms associated with upregulated and downregulated transcripts depicted in . GO-term analysis was performed with DAVID .

    Techniques Used: Activation Assay, Binding Assay, Inhibition, Genome Wide, ChIP-sequencing, RNA Sequencing Assay

    ( A ) Genome browser screenshots of representative de-repressed genes upon PRC1 inhibition. Both six3b and six2b are bound by Rnf2 and marked by H2Aub1 from prezygotic genome activation (preZGA) onward. At postZGA these genes normally attract binding by Aebp2 and acquire H3K27me3. Inhibition of Rnf2 (via PRT4165) causes failed Aebp2 binding, and prevention of H3K27me3 establishment. Notably, six3b is precociously expressed upon loss of H2Aub1. ( B ) Genome browser screenshots of representative loci that gain ectopic Aebp2 binding (left) or H3K27me3 marking (right) upon PRC1 inhibition. Ectopic binding events for Aebp2 or H3K27me3 appear to be mutually exclusive. Dotted boxes denote region of ectopic binding or marking by Aebp2 or H3K27me3, respectively. ( C ) Approximately 16.6% of genes normally silenced by H2Aub1 promoter marking are transcriptionally upregulated upon inhibition of Rnf2. Venn diagram displays the overlap between the gene promoters normally marked by H2Aub1 and protein coding genes that are transcriptionally upregulated (p-value < 0.001, >1.5-fold change) after Rnf2 inhibition. Here, H2Aub1 marked promoters are defined as regions ±2 Kb from transcription start sites and having peak(s) called by MACS2 (q-value cutoff 0.01) of H2Aub1 from 4.3 hpf H2Aub1 ChIP-seq.
    Figure Legend Snippet: ( A ) Genome browser screenshots of representative de-repressed genes upon PRC1 inhibition. Both six3b and six2b are bound by Rnf2 and marked by H2Aub1 from prezygotic genome activation (preZGA) onward. At postZGA these genes normally attract binding by Aebp2 and acquire H3K27me3. Inhibition of Rnf2 (via PRT4165) causes failed Aebp2 binding, and prevention of H3K27me3 establishment. Notably, six3b is precociously expressed upon loss of H2Aub1. ( B ) Genome browser screenshots of representative loci that gain ectopic Aebp2 binding (left) or H3K27me3 marking (right) upon PRC1 inhibition. Ectopic binding events for Aebp2 or H3K27me3 appear to be mutually exclusive. Dotted boxes denote region of ectopic binding or marking by Aebp2 or H3K27me3, respectively. ( C ) Approximately 16.6% of genes normally silenced by H2Aub1 promoter marking are transcriptionally upregulated upon inhibition of Rnf2. Venn diagram displays the overlap between the gene promoters normally marked by H2Aub1 and protein coding genes that are transcriptionally upregulated (p-value < 0.001, >1.5-fold change) after Rnf2 inhibition. Here, H2Aub1 marked promoters are defined as regions ±2 Kb from transcription start sites and having peak(s) called by MACS2 (q-value cutoff 0.01) of H2Aub1 from 4.3 hpf H2Aub1 ChIP-seq.

    Techniques Used: Inhibition, Activation Assay, Binding Assay, ChIP-sequencing

    Prior to zygotic genome activation (ZGA), Rnf2-PRC1 is recruited by transcription factors (TFs) to promoters (shown) and enhancers (not shown) of developmental genes bearing Placeholder nucleosomes (H2A.Z(FV), H3K4me1, H3K27ac). Rnf2-PRC1 deposition of H2Aub1 recruits Aebp2-PRC2 to catalyze H3K27me3 addition. H2Aub1 ablation (via Rnf2 inhibition) eliminates Aebp2-PRC2 recruitment and prevents H3K27me3 establishment. Notably, H2Aub1 loss causes precocious transcription of certain developmental genes after ZGA, identifying H2Aub1 as a critical component of silencing at ZGA.
    Figure Legend Snippet: Prior to zygotic genome activation (ZGA), Rnf2-PRC1 is recruited by transcription factors (TFs) to promoters (shown) and enhancers (not shown) of developmental genes bearing Placeholder nucleosomes (H2A.Z(FV), H3K4me1, H3K27ac). Rnf2-PRC1 deposition of H2Aub1 recruits Aebp2-PRC2 to catalyze H3K27me3 addition. H2Aub1 ablation (via Rnf2 inhibition) eliminates Aebp2-PRC2 recruitment and prevents H3K27me3 establishment. Notably, H2Aub1 loss causes precocious transcription of certain developmental genes after ZGA, identifying H2Aub1 as a critical component of silencing at ZGA.

    Techniques Used: Activation Assay, Inhibition


    Figure Legend Snippet:

    Techniques Used: Software

    rabbit monoclonal anti aebp2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit monoclonal anti aebp2
    a Violin plots showing the average expression levels of components of PRC2 in the Mix, Proxi, and Dista clusters. To increase the reproducibility, the gene expressions of H3K27me3 and H3K4me3 scSET-seq were merged for plotting. P values were calculated by Student’s t -test, two-sided. Rbbp7 was not detected in the scSET-seq. n = 291 (Mix), 133 (Proxi), and 121 (Dista) cells. b Western blot showing the indicated protein levels in parental and KO mESCs. Ezh2 KO decreased the total levels of H3K27me3 and increased the H3K27ac. <t>Aebp2</t> KO didn’t exhibit obvious effects on the total levels of tested histone marks. Cell extracts were analyzed via Western blotting using the indicated antibodies, and two biological replicates were performed for each blot. Star indicated a nonspecific band. See Supplementary Fig. for the Sanger sequencing results of KO clones. c Representative images of divided Nanog - mCherry mESCs cocultured with Wnt3a beads. Wnt3a beads and cells were marked in the immunofluorescence image. Asymmetric, higher mCherry amounts in the bead-proximal cell; Symmetric, similar amounts of mCherry in either cell; Reverse, higher amounts of mCherry in the bead-distal cell. BF, bright field. Scale bar, 15 µm. d The percentages of asymmetrically, symmetrically, and reversely divided cells, as defined by Nanog - mCherry signals, in parental, Ezh2 and Aebp2 KO cells. P values were calculated by chi-squared test. e The enrichments of AEBP2 at cluster marker genes that were identified by gene expression. AEBP2 peaks were first called and annotated to the closet transcription starting sites within 20 Kb. The reads density of Aebp2 was then calculated for peaks annotated to each gene. P values were calculated by Student’s t -test, two-sided. The boxes were drawn from lower quartile (Q1) to upper quartile (Q3) with the middle line denoting the median, and whiskers with maximum 1.5 IQR (interquartile range). n = 90, 108, and 200 Aebp2 peaks at Mix, Proxi and Dista markers, respectively. n = 1000 (Mix), 1000 (Proxi), and 1000 (Dista) bins of Aebp2 at cluster marker genes. f The enrichments of H3K27me3 at cluster marker genes that were identified by gene expression. H3K27me3 peaks were first called in WT and Aebp2 KO cells, merged, and annotated to the closet transcription starting sites within 20 Kb. The reads density of H3K27me3 was then calculated for peaks annotated to each gene. P values were calculated by Student’s t-test, two-sided. The boxes were drawn from lower quartile (Q1) to upper quartile (Q3) with the middle line denoting the median, and whiskers with maximum 1.5 IQR (interquartile range). n = 504, 243, and 371 H3K27me3 peaks at Mix, Proxi, and Dista markers, respectively. Source data are provided as a Source Data file.
    Rabbit Monoclonal Anti Aebp2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
    rabbit monoclonal anti aebp2 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Joint single-cell multiomic analysis in Wnt3a induced asymmetric stem cell division"

    Article Title: Joint single-cell multiomic analysis in Wnt3a induced asymmetric stem cell division

    Journal: Nature Communications

    doi: 10.1038/s41467-021-26203-0

    a Violin plots showing the average expression levels of components of PRC2 in the Mix, Proxi, and Dista clusters. To increase the reproducibility, the gene expressions of H3K27me3 and H3K4me3 scSET-seq were merged for plotting. P values were calculated by Student’s t -test, two-sided. Rbbp7 was not detected in the scSET-seq. n = 291 (Mix), 133 (Proxi), and 121 (Dista) cells. b Western blot showing the indicated protein levels in parental and KO mESCs. Ezh2 KO decreased the total levels of H3K27me3 and increased the H3K27ac. Aebp2 KO didn’t exhibit obvious effects on the total levels of tested histone marks. Cell extracts were analyzed via Western blotting using the indicated antibodies, and two biological replicates were performed for each blot. Star indicated a nonspecific band. See Supplementary Fig. for the Sanger sequencing results of KO clones. c Representative images of divided Nanog - mCherry mESCs cocultured with Wnt3a beads. Wnt3a beads and cells were marked in the immunofluorescence image. Asymmetric, higher mCherry amounts in the bead-proximal cell; Symmetric, similar amounts of mCherry in either cell; Reverse, higher amounts of mCherry in the bead-distal cell. BF, bright field. Scale bar, 15 µm. d The percentages of asymmetrically, symmetrically, and reversely divided cells, as defined by Nanog - mCherry signals, in parental, Ezh2 and Aebp2 KO cells. P values were calculated by chi-squared test. e The enrichments of AEBP2 at cluster marker genes that were identified by gene expression. AEBP2 peaks were first called and annotated to the closet transcription starting sites within 20 Kb. The reads density of Aebp2 was then calculated for peaks annotated to each gene. P values were calculated by Student’s t -test, two-sided. The boxes were drawn from lower quartile (Q1) to upper quartile (Q3) with the middle line denoting the median, and whiskers with maximum 1.5 IQR (interquartile range). n = 90, 108, and 200 Aebp2 peaks at Mix, Proxi and Dista markers, respectively. n = 1000 (Mix), 1000 (Proxi), and 1000 (Dista) bins of Aebp2 at cluster marker genes. f The enrichments of H3K27me3 at cluster marker genes that were identified by gene expression. H3K27me3 peaks were first called in WT and Aebp2 KO cells, merged, and annotated to the closet transcription starting sites within 20 Kb. The reads density of H3K27me3 was then calculated for peaks annotated to each gene. P values were calculated by Student’s t-test, two-sided. The boxes were drawn from lower quartile (Q1) to upper quartile (Q3) with the middle line denoting the median, and whiskers with maximum 1.5 IQR (interquartile range). n = 504, 243, and 371 H3K27me3 peaks at Mix, Proxi, and Dista markers, respectively. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Violin plots showing the average expression levels of components of PRC2 in the Mix, Proxi, and Dista clusters. To increase the reproducibility, the gene expressions of H3K27me3 and H3K4me3 scSET-seq were merged for plotting. P values were calculated by Student’s t -test, two-sided. Rbbp7 was not detected in the scSET-seq. n = 291 (Mix), 133 (Proxi), and 121 (Dista) cells. b Western blot showing the indicated protein levels in parental and KO mESCs. Ezh2 KO decreased the total levels of H3K27me3 and increased the H3K27ac. Aebp2 KO didn’t exhibit obvious effects on the total levels of tested histone marks. Cell extracts were analyzed via Western blotting using the indicated antibodies, and two biological replicates were performed for each blot. Star indicated a nonspecific band. See Supplementary Fig. for the Sanger sequencing results of KO clones. c Representative images of divided Nanog - mCherry mESCs cocultured with Wnt3a beads. Wnt3a beads and cells were marked in the immunofluorescence image. Asymmetric, higher mCherry amounts in the bead-proximal cell; Symmetric, similar amounts of mCherry in either cell; Reverse, higher amounts of mCherry in the bead-distal cell. BF, bright field. Scale bar, 15 µm. d The percentages of asymmetrically, symmetrically, and reversely divided cells, as defined by Nanog - mCherry signals, in parental, Ezh2 and Aebp2 KO cells. P values were calculated by chi-squared test. e The enrichments of AEBP2 at cluster marker genes that were identified by gene expression. AEBP2 peaks were first called and annotated to the closet transcription starting sites within 20 Kb. The reads density of Aebp2 was then calculated for peaks annotated to each gene. P values were calculated by Student’s t -test, two-sided. The boxes were drawn from lower quartile (Q1) to upper quartile (Q3) with the middle line denoting the median, and whiskers with maximum 1.5 IQR (interquartile range). n = 90, 108, and 200 Aebp2 peaks at Mix, Proxi and Dista markers, respectively. n = 1000 (Mix), 1000 (Proxi), and 1000 (Dista) bins of Aebp2 at cluster marker genes. f The enrichments of H3K27me3 at cluster marker genes that were identified by gene expression. H3K27me3 peaks were first called in WT and Aebp2 KO cells, merged, and annotated to the closet transcription starting sites within 20 Kb. The reads density of H3K27me3 was then calculated for peaks annotated to each gene. P values were calculated by Student’s t-test, two-sided. The boxes were drawn from lower quartile (Q1) to upper quartile (Q3) with the middle line denoting the median, and whiskers with maximum 1.5 IQR (interquartile range). n = 504, 243, and 371 H3K27me3 peaks at Mix, Proxi, and Dista markers, respectively. Source data are provided as a Source Data file.

    Techniques Used: Expressing, Western Blot, Sequencing, Clone Assay, Immunofluorescence, Marker

    rabbit monoclonal anti aebp2 d7c6x  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit monoclonal anti aebp2 d7c6x
    PRC2 sub-complexes differentially contribute to the BAP1-dependent intergenic H3K27me3 deposition (A) Cartoon showing divergent compositions of the PRC2.1 and PRC2.2 complexes and their differing affinities for chromatin features. (B) Western blot analysis with the indicated antibodies on total protein extracts from the Bap1 KO, <t>Aebp2</t> + Jarid2 ( A+J ) KO, Aebp2 + Jarid2 + Bap1 ( A+J+B ) KO, and matching WT ESCs. (C) Western blot analysis with the indicated antibodies on total protein extracts from the indicated ESC lines. (D) Boxplots representing H2AK119ub1 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (E) Boxplots representing H3K27me3 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (F) Boxplots showing the log2 fold change RPKM ratio for H2AK119ub1 or H3K27me3 in the indicated cell lines (A = Aebp2 , J = Jarid2 , P = Pcl1-3 ) at intergenic regions (n = 38,068). (G) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Aebp2/Jarid2/Bap1 KO versus Aebp2/Jarid2 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. (H) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Pcl1-3/Bap1 KO versus Pcl1-3 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. See also <xref ref-type=Figure S4 . " width="250" height="auto" />
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    Images

    1) Product Images from "BAP1 enhances Polycomb repression by counteracting widespread H2AK119ub1 deposition and chromatin condensation"

    Article Title: BAP1 enhances Polycomb repression by counteracting widespread H2AK119ub1 deposition and chromatin condensation

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2021.06.020

    PRC2 sub-complexes differentially contribute to the BAP1-dependent intergenic H3K27me3 deposition (A) Cartoon showing divergent compositions of the PRC2.1 and PRC2.2 complexes and their differing affinities for chromatin features. (B) Western blot analysis with the indicated antibodies on total protein extracts from the Bap1 KO, Aebp2 + Jarid2 ( A+J ) KO, Aebp2 + Jarid2 + Bap1 ( A+J+B ) KO, and matching WT ESCs. (C) Western blot analysis with the indicated antibodies on total protein extracts from the indicated ESC lines. (D) Boxplots representing H2AK119ub1 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (E) Boxplots representing H3K27me3 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (F) Boxplots showing the log2 fold change RPKM ratio for H2AK119ub1 or H3K27me3 in the indicated cell lines (A = Aebp2 , J = Jarid2 , P = Pcl1-3 ) at intergenic regions (n = 38,068). (G) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Aebp2/Jarid2/Bap1 KO versus Aebp2/Jarid2 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. (H) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Pcl1-3/Bap1 KO versus Pcl1-3 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. See also <xref ref-type=Figure S4 . " title="... on total protein extracts from the Bap1 KO, Aebp2 + Jarid2 ( A+J ) KO, Aebp2 + ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: PRC2 sub-complexes differentially contribute to the BAP1-dependent intergenic H3K27me3 deposition (A) Cartoon showing divergent compositions of the PRC2.1 and PRC2.2 complexes and their differing affinities for chromatin features. (B) Western blot analysis with the indicated antibodies on total protein extracts from the Bap1 KO, Aebp2 + Jarid2 ( A+J ) KO, Aebp2 + Jarid2 + Bap1 ( A+J+B ) KO, and matching WT ESCs. (C) Western blot analysis with the indicated antibodies on total protein extracts from the indicated ESC lines. (D) Boxplots representing H2AK119ub1 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (E) Boxplots representing H3K27me3 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (F) Boxplots showing the log2 fold change RPKM ratio for H2AK119ub1 or H3K27me3 in the indicated cell lines (A = Aebp2 , J = Jarid2 , P = Pcl1-3 ) at intergenic regions (n = 38,068). (G) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Aebp2/Jarid2/Bap1 KO versus Aebp2/Jarid2 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. (H) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Pcl1-3/Bap1 KO versus Pcl1-3 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. See also Figure S4 .

    Techniques Used: Western Blot, ChIP-sequencing, Genome Wide


    Figure Legend Snippet:

    Techniques Used: Recombinant, Transfection, Purification, Sequencing, Imaging, Plasmid Preparation, Software

    anti aebp2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti aebp2
    PRC2 sub-complexes differentially contribute to the BAP1-dependent intergenic H3K27me3 deposition (A) Cartoon showing divergent compositions of the PRC2.1 and PRC2.2 complexes and their differing affinities for chromatin features. (B) Western blot analysis with the indicated antibodies on total protein extracts from the Bap1 KO, <t>Aebp2</t> + Jarid2 ( A+J ) KO, Aebp2 + Jarid2 + Bap1 ( A+J+B ) KO, and matching WT ESCs. (C) Western blot analysis with the indicated antibodies on total protein extracts from the indicated ESC lines. (D) Boxplots representing H2AK119ub1 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (E) Boxplots representing H3K27me3 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (F) Boxplots showing the log2 fold change RPKM ratio for H2AK119ub1 or H3K27me3 in the indicated cell lines (A = Aebp2 , J = Jarid2 , P = Pcl1-3 ) at intergenic regions (n = 38,068). (G) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Aebp2/Jarid2/Bap1 KO versus Aebp2/Jarid2 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. (H) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Pcl1-3/Bap1 KO versus Pcl1-3 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. See also <xref ref-type=Figure S4 . " width="250" height="auto" />
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    Images

    1) Product Images from "BAP1 enhances Polycomb repression by counteracting widespread H2AK119ub1 deposition and chromatin condensation"

    Article Title: BAP1 enhances Polycomb repression by counteracting widespread H2AK119ub1 deposition and chromatin condensation

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2021.06.020

    PRC2 sub-complexes differentially contribute to the BAP1-dependent intergenic H3K27me3 deposition (A) Cartoon showing divergent compositions of the PRC2.1 and PRC2.2 complexes and their differing affinities for chromatin features. (B) Western blot analysis with the indicated antibodies on total protein extracts from the Bap1 KO, Aebp2 + Jarid2 ( A+J ) KO, Aebp2 + Jarid2 + Bap1 ( A+J+B ) KO, and matching WT ESCs. (C) Western blot analysis with the indicated antibodies on total protein extracts from the indicated ESC lines. (D) Boxplots representing H2AK119ub1 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (E) Boxplots representing H3K27me3 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (F) Boxplots showing the log2 fold change RPKM ratio for H2AK119ub1 or H3K27me3 in the indicated cell lines (A = Aebp2 , J = Jarid2 , P = Pcl1-3 ) at intergenic regions (n = 38,068). (G) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Aebp2/Jarid2/Bap1 KO versus Aebp2/Jarid2 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. (H) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Pcl1-3/Bap1 KO versus Pcl1-3 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. See also <xref ref-type=Figure S4 . " title="... on total protein extracts from the Bap1 KO, Aebp2 + Jarid2 ( A+J ) KO, Aebp2 + ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: PRC2 sub-complexes differentially contribute to the BAP1-dependent intergenic H3K27me3 deposition (A) Cartoon showing divergent compositions of the PRC2.1 and PRC2.2 complexes and their differing affinities for chromatin features. (B) Western blot analysis with the indicated antibodies on total protein extracts from the Bap1 KO, Aebp2 + Jarid2 ( A+J ) KO, Aebp2 + Jarid2 + Bap1 ( A+J+B ) KO, and matching WT ESCs. (C) Western blot analysis with the indicated antibodies on total protein extracts from the indicated ESC lines. (D) Boxplots representing H2AK119ub1 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (E) Boxplots representing H3K27me3 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (F) Boxplots showing the log2 fold change RPKM ratio for H2AK119ub1 or H3K27me3 in the indicated cell lines (A = Aebp2 , J = Jarid2 , P = Pcl1-3 ) at intergenic regions (n = 38,068). (G) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Aebp2/Jarid2/Bap1 KO versus Aebp2/Jarid2 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. (H) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Pcl1-3/Bap1 KO versus Pcl1-3 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. See also Figure S4 .

    Techniques Used: Western Blot, ChIP-sequencing, Genome Wide


    Figure Legend Snippet:

    Techniques Used: Recombinant, Transfection, Purification, Sequencing, Imaging, Plasmid Preparation, Software

    aebp2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc aebp2
    a. Models of SUZ12, SUZ12Δ93, JAZF1-SUZ12 and JAZF1 constructs with C-terminal FLAG tags (F). b. Immunoblots for FLAG, SUZ12 (N-terminus), EZH2, JARID2, <t>AEBP2,</t> PCL2, EPOP, TRRAP and β-actin in input (In.) and SUZ12 IPs from WT E14 cells or input and FLAG IPs from Suz12 GT/GT cells stably expressing FLAG-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12 or JAZF1. c. Immunoblots for FLAG, HA-PALI and EZH2 in input and Strep-Tactin pull-downs (P) from NIH-3T3 cells transfected with HA-tagged PALI1 and FS2-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12 or JAZF1. d. See also .
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    1) Product Images from "JAZF1-SUZ12 dysregulates PRC2 function and gene expression during cell differentiation"

    Article Title: JAZF1-SUZ12 dysregulates PRC2 function and gene expression during cell differentiation

    Journal: bioRxiv

    doi: 10.1101/2021.04.15.440052

    a. Models of SUZ12, SUZ12Δ93, JAZF1-SUZ12 and JAZF1 constructs with C-terminal FLAG tags (F). b. Immunoblots for FLAG, SUZ12 (N-terminus), EZH2, JARID2, AEBP2, PCL2, EPOP, TRRAP and β-actin in input (In.) and SUZ12 IPs from WT E14 cells or input and FLAG IPs from Suz12 GT/GT cells stably expressing FLAG-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12 or JAZF1. c. Immunoblots for FLAG, HA-PALI and EZH2 in input and Strep-Tactin pull-downs (P) from NIH-3T3 cells transfected with HA-tagged PALI1 and FS2-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12 or JAZF1. d. See also .
    Figure Legend Snippet: a. Models of SUZ12, SUZ12Δ93, JAZF1-SUZ12 and JAZF1 constructs with C-terminal FLAG tags (F). b. Immunoblots for FLAG, SUZ12 (N-terminus), EZH2, JARID2, AEBP2, PCL2, EPOP, TRRAP and β-actin in input (In.) and SUZ12 IPs from WT E14 cells or input and FLAG IPs from Suz12 GT/GT cells stably expressing FLAG-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12 or JAZF1. c. Immunoblots for FLAG, HA-PALI and EZH2 in input and Strep-Tactin pull-downs (P) from NIH-3T3 cells transfected with HA-tagged PALI1 and FS2-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12 or JAZF1. d. See also .

    Techniques Used: Construct, Western Blot, Stable Transfection, Expressing, Transfection

    a. Levels of FLAG-tagged GFP, SUZ12 (2 different clones), SUZ12Δ93, JAZF1-SUZ12 and JAZF1 in stable Suz12 GT/GT cell lines in comparison to WT E14 cells. b. Immunoblots for V5 and HA in input (In.) and SUZ12 IP fractions from NIH-3T3 cells co-transfected with V5-tagged PCL3 and HA-tagged SUZ12/JAZF1 constructs. c. As b., except in cells transfected with FS2-EPOP and HA-tagged SUZ12/JAZF1 constructs. d. As b., except in cells transfected with FS2-AEBP2 and HA-tagged SUZ12/JAZF1 constructs.
    Figure Legend Snippet: a. Levels of FLAG-tagged GFP, SUZ12 (2 different clones), SUZ12Δ93, JAZF1-SUZ12 and JAZF1 in stable Suz12 GT/GT cell lines in comparison to WT E14 cells. b. Immunoblots for V5 and HA in input (In.) and SUZ12 IP fractions from NIH-3T3 cells co-transfected with V5-tagged PCL3 and HA-tagged SUZ12/JAZF1 constructs. c. As b., except in cells transfected with FS2-EPOP and HA-tagged SUZ12/JAZF1 constructs. d. As b., except in cells transfected with FS2-AEBP2 and HA-tagged SUZ12/JAZF1 constructs.

    Techniques Used: Clone Assay, Western Blot, Transfection, Construct

    a. Left: Immunoblots for FLAG, EZH2, FUS, β-actin and H3 in nucleoplasm (Np) and chromatin (Ch) fractions from mock or RNaseA-treated Suz12 GT/GT cells expressing either FLAG-tagged SUZ12, SUZ12Δ93 or JAZF1-SUZ12. Representative of three independent experiments. Right : Fold change in FLAG, EZH2, FUS and β-actin in the chromatin fraction upon RNaseA treatment (mean and s.d, n=3). b. As a., but for endogenous SUZ12, EZH2, JARID2, FUS, β-actin and H3 in Aebp2 WT/WT , Aebp2 GT/GT and Jarid2 GT/GT ESC. c. As a., but for endogenous SUZ12, EZH2, EPOP, FUS, β-actin and H3 in Epop WT/WT and Epop GT/GT ESC. See also .
    Figure Legend Snippet: a. Left: Immunoblots for FLAG, EZH2, FUS, β-actin and H3 in nucleoplasm (Np) and chromatin (Ch) fractions from mock or RNaseA-treated Suz12 GT/GT cells expressing either FLAG-tagged SUZ12, SUZ12Δ93 or JAZF1-SUZ12. Representative of three independent experiments. Right : Fold change in FLAG, EZH2, FUS and β-actin in the chromatin fraction upon RNaseA treatment (mean and s.d, n=3). b. As a., but for endogenous SUZ12, EZH2, JARID2, FUS, β-actin and H3 in Aebp2 WT/WT , Aebp2 GT/GT and Jarid2 GT/GT ESC. c. As a., but for endogenous SUZ12, EZH2, EPOP, FUS, β-actin and H3 in Epop WT/WT and Epop GT/GT ESC. See also .

    Techniques Used: Western Blot, Expressing

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    Cell Signaling Technology Inc aebp2
    JAZF1-SUZ12 lacks interaction with JARID2, EPOP, and PALI1 due to loss of the SUZ12 N terminus (A) Models of FLAG-tagged (F) SUZ12, SUZ12Δ93, JAZF1-SUZ12, and JAZF1 constructs. (B) Immunoblots for FLAG, SUZ12 (N terminus), EZH2, JARID2, <t>AEBP2,</t> PCL2, EPOP, TRRAP, and β-actin in input (In.) and SUZ12 IPs from WT or Suz12 GT/GT ESCs stably expressing FLAG-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent IPs. (C) Immunoblots for FLAG, HA-PALI, and EZH2 in input and Strep-Tactin pull-downs (P) from NIH3T3 cells transfected with HA-PALI1 and FS2-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent pull-downs. See also <xref ref-type=Figure S1 . " width="250" height="auto" />
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    Cell Signaling Technology Inc anti aebp2
    (A) Timeline of GapmeR-mediated gene knockdown and dox-induced Firre transgene expression. (B) Depiction of possible outcomes for Firre -responsive gene expression (red line) after knockdown of a regulatory factor (blue line) and induction of Firre transgene expression. (C) Relative gene expression of Firre and its targets Adgrg1 and Shf in Firre OE cells transfected with non-targeting control-(NTC), Ezh2 -, Suz12, Jarid2 -, <t>Aebp2</t> -, Wdr5 -, or G9a -targeting GapmeRs, and treated without (zero hours) or with (six hours) dox as determined by RT-qPCR. (D and E) smRNA FISH-based quantification of the distance between Shf intron and Firre transgene (D) and Shf intron and the five closest Firre exons (distance averaged) (E) in Firre -overexpression and Firre -rescue cells after 0, 2, 4, and 6 hours of dox treatment. The images below depict the rationale of the distance measurements.
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    E2f2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc s rrid ab 2798398
    PCR primer sequences
    S Rrid Ab 2798398, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/s rrid ab 2798398/product/Cell Signaling Technology Inc
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    Cell Signaling Technology Inc rabbit monoclonal anti aebp2
    a Violin plots showing the average expression levels of components of PRC2 in the Mix, Proxi, and Dista clusters. To increase the reproducibility, the gene expressions of H3K27me3 and H3K4me3 scSET-seq were merged for plotting. P values were calculated by Student’s t -test, two-sided. Rbbp7 was not detected in the scSET-seq. n = 291 (Mix), 133 (Proxi), and 121 (Dista) cells. b Western blot showing the indicated protein levels in parental and KO mESCs. Ezh2 KO decreased the total levels of H3K27me3 and increased the H3K27ac. <t>Aebp2</t> KO didn’t exhibit obvious effects on the total levels of tested histone marks. Cell extracts were analyzed via Western blotting using the indicated antibodies, and two biological replicates were performed for each blot. Star indicated a nonspecific band. See Supplementary Fig. for the Sanger sequencing results of KO clones. c Representative images of divided Nanog - mCherry mESCs cocultured with Wnt3a beads. Wnt3a beads and cells were marked in the immunofluorescence image. Asymmetric, higher mCherry amounts in the bead-proximal cell; Symmetric, similar amounts of mCherry in either cell; Reverse, higher amounts of mCherry in the bead-distal cell. BF, bright field. Scale bar, 15 µm. d The percentages of asymmetrically, symmetrically, and reversely divided cells, as defined by Nanog - mCherry signals, in parental, Ezh2 and Aebp2 KO cells. P values were calculated by chi-squared test. e The enrichments of AEBP2 at cluster marker genes that were identified by gene expression. AEBP2 peaks were first called and annotated to the closet transcription starting sites within 20 Kb. The reads density of Aebp2 was then calculated for peaks annotated to each gene. P values were calculated by Student’s t -test, two-sided. The boxes were drawn from lower quartile (Q1) to upper quartile (Q3) with the middle line denoting the median, and whiskers with maximum 1.5 IQR (interquartile range). n = 90, 108, and 200 Aebp2 peaks at Mix, Proxi and Dista markers, respectively. n = 1000 (Mix), 1000 (Proxi), and 1000 (Dista) bins of Aebp2 at cluster marker genes. f The enrichments of H3K27me3 at cluster marker genes that were identified by gene expression. H3K27me3 peaks were first called in WT and Aebp2 KO cells, merged, and annotated to the closet transcription starting sites within 20 Kb. The reads density of H3K27me3 was then calculated for peaks annotated to each gene. P values were calculated by Student’s t-test, two-sided. The boxes were drawn from lower quartile (Q1) to upper quartile (Q3) with the middle line denoting the median, and whiskers with maximum 1.5 IQR (interquartile range). n = 504, 243, and 371 H3K27me3 peaks at Mix, Proxi, and Dista markers, respectively. Source data are provided as a Source Data file.
    Rabbit Monoclonal Anti Aebp2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit monoclonal anti aebp2 d7c6x
    PRC2 sub-complexes differentially contribute to the BAP1-dependent intergenic H3K27me3 deposition (A) Cartoon showing divergent compositions of the PRC2.1 and PRC2.2 complexes and their differing affinities for chromatin features. (B) Western blot analysis with the indicated antibodies on total protein extracts from the Bap1 KO, <t>Aebp2</t> + Jarid2 ( A+J ) KO, Aebp2 + Jarid2 + Bap1 ( A+J+B ) KO, and matching WT ESCs. (C) Western blot analysis with the indicated antibodies on total protein extracts from the indicated ESC lines. (D) Boxplots representing H2AK119ub1 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (E) Boxplots representing H3K27me3 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (F) Boxplots showing the log2 fold change RPKM ratio for H2AK119ub1 or H3K27me3 in the indicated cell lines (A = Aebp2 , J = Jarid2 , P = Pcl1-3 ) at intergenic regions (n = 38,068). (G) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Aebp2/Jarid2/Bap1 KO versus Aebp2/Jarid2 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. (H) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Pcl1-3/Bap1 KO versus Pcl1-3 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. See also <xref ref-type=Figure S4 . " width="250" height="auto" />
    Rabbit Monoclonal Anti Aebp2 D7c6x, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    JAZF1-SUZ12 lacks interaction with JARID2, EPOP, and PALI1 due to loss of the SUZ12 N terminus (A) Models of FLAG-tagged (F) SUZ12, SUZ12Δ93, JAZF1-SUZ12, and JAZF1 constructs. (B) Immunoblots for FLAG, SUZ12 (N terminus), EZH2, JARID2, AEBP2, PCL2, EPOP, TRRAP, and β-actin in input (In.) and SUZ12 IPs from WT or Suz12 GT/GT ESCs stably expressing FLAG-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent IPs. (C) Immunoblots for FLAG, HA-PALI, and EZH2 in input and Strep-Tactin pull-downs (P) from NIH3T3 cells transfected with HA-PALI1 and FS2-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent pull-downs. See also <xref ref-type=Figure S1 . " width="100%" height="100%">

    Journal: Cell Reports

    Article Title: JAZF1-SUZ12 dysregulates PRC2 function and gene expression during cell differentiation

    doi: 10.1016/j.celrep.2022.110889

    Figure Lengend Snippet: JAZF1-SUZ12 lacks interaction with JARID2, EPOP, and PALI1 due to loss of the SUZ12 N terminus (A) Models of FLAG-tagged (F) SUZ12, SUZ12Δ93, JAZF1-SUZ12, and JAZF1 constructs. (B) Immunoblots for FLAG, SUZ12 (N terminus), EZH2, JARID2, AEBP2, PCL2, EPOP, TRRAP, and β-actin in input (In.) and SUZ12 IPs from WT or Suz12 GT/GT ESCs stably expressing FLAG-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent IPs. (C) Immunoblots for FLAG, HA-PALI, and EZH2 in input and Strep-Tactin pull-downs (P) from NIH3T3 cells transfected with HA-PALI1 and FS2-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent pull-downs. See also Figure S1 .

    Article Snippet: Proteins were detected with primary antibodies to FLAG M2 (Sigma, A8592), HA 3F10 (Roche, 12013819001), V5 (Abcam, ab15828), SUZ12 (Santa Cruz sc-46264), EZH2 (CST 3147), JARID2 (CST 13594), AEBP2 (CST 14129), PCL2 (Proteintech 16208-1-AP), EPOP (kind gift of L. Di Croce), TRRAP (Abcam, ab73546), FUS (Novus Biologicals 100–565), β-actin (CST 4967), alpha tubulin (CST 2144), H3K27me3 (Abcam ab192985), H4 pan-acetyl (Thermofisher, PA5-40083) and H3 (Abcam ab1791) and HRP-conjugated secondary antibodies (anti-mouse (Dako, P0447) or anti-rabbit (Dako, P0448).

    Techniques: Construct, Western Blot, Stable Transfection, Expressing, Transfection

    Journal: Cell Reports

    Article Title: JAZF1-SUZ12 dysregulates PRC2 function and gene expression during cell differentiation

    doi: 10.1016/j.celrep.2022.110889

    Figure Lengend Snippet:

    Article Snippet: Proteins were detected with primary antibodies to FLAG M2 (Sigma, A8592), HA 3F10 (Roche, 12013819001), V5 (Abcam, ab15828), SUZ12 (Santa Cruz sc-46264), EZH2 (CST 3147), JARID2 (CST 13594), AEBP2 (CST 14129), PCL2 (Proteintech 16208-1-AP), EPOP (kind gift of L. Di Croce), TRRAP (Abcam, ab73546), FUS (Novus Biologicals 100–565), β-actin (CST 4967), alpha tubulin (CST 2144), H3K27me3 (Abcam ab192985), H4 pan-acetyl (Thermofisher, PA5-40083) and H3 (Abcam ab1791) and HRP-conjugated secondary antibodies (anti-mouse (Dako, P0447) or anti-rabbit (Dako, P0448).

    Techniques: Recombinant, Avidin-Biotin Assay, SYBR Green Assay, Sensitive Assay, Western Blot, Software

    (A) Timeline of GapmeR-mediated gene knockdown and dox-induced Firre transgene expression. (B) Depiction of possible outcomes for Firre -responsive gene expression (red line) after knockdown of a regulatory factor (blue line) and induction of Firre transgene expression. (C) Relative gene expression of Firre and its targets Adgrg1 and Shf in Firre OE cells transfected with non-targeting control-(NTC), Ezh2 -, Suz12, Jarid2 -, Aebp2 -, Wdr5 -, or G9a -targeting GapmeRs, and treated without (zero hours) or with (six hours) dox as determined by RT-qPCR. (D and E) smRNA FISH-based quantification of the distance between Shf intron and Firre transgene (D) and Shf intron and the five closest Firre exons (distance averaged) (E) in Firre -overexpression and Firre -rescue cells after 0, 2, 4, and 6 hours of dox treatment. The images below depict the rationale of the distance measurements.

    Journal: bioRxiv

    Article Title: The lncRNA Firre functions as a transcriptional activator from a distance

    doi: 10.1101/2022.05.15.492001

    Figure Lengend Snippet: (A) Timeline of GapmeR-mediated gene knockdown and dox-induced Firre transgene expression. (B) Depiction of possible outcomes for Firre -responsive gene expression (red line) after knockdown of a regulatory factor (blue line) and induction of Firre transgene expression. (C) Relative gene expression of Firre and its targets Adgrg1 and Shf in Firre OE cells transfected with non-targeting control-(NTC), Ezh2 -, Suz12, Jarid2 -, Aebp2 -, Wdr5 -, or G9a -targeting GapmeRs, and treated without (zero hours) or with (six hours) dox as determined by RT-qPCR. (D and E) smRNA FISH-based quantification of the distance between Shf intron and Firre transgene (D) and Shf intron and the five closest Firre exons (distance averaged) (E) in Firre -overexpression and Firre -rescue cells after 0, 2, 4, and 6 hours of dox treatment. The images below depict the rationale of the distance measurements.

    Article Snippet: Membranes were blocked in 5% milk in TBS-T (TBS with 0.1% Tween 20) and probed with anti-JARID2 (D6M9X), anti-AEBP2 (D7C6X), anti-EZH2 (D2C9), anti-SUZ12 (Cell Signaling Technology, D39F6), or anti-GAPDH antibody (14C10, all Cell Signaling Technology) at a dilution of 1:1000 in blocking buffer overnight at 4°C or for one hour at room temperature.

    Techniques: Expressing, Transfection, Quantitative RT-PCR, Over Expression

    PCR primer sequences

    Journal: Bioengineered

    Article Title: MicroRNA-631 deriving from bone marrow mesenchymal stem cell exosomes facilitates the malignant behavior of non-small cell lung cancer via modulating the E2F family of transcription factor 2/phosphatidylinositol 3‐kinase/Akt signaling pathway

    doi: 10.1080/21655979.2022.2036891

    Figure Lengend Snippet: PCR primer sequences

    Article Snippet: A total of 20 g samples were applied to 12% sulfate-polyacrylamide gel, electrophoresis was performed to separate proteins, and then electroblot was onto polyvinylidene fluoride membrane (Invitrogen, Carlsbad, CA, USA), which was sealed with 5% skimmed milk powder for 1 h. Subsequently, incubation of the membrane was with the following primary antibodies: CD9 (ab92726, Abcam), E2F2 (14,129, 1:1000, Cell Signaling Technology), CD81 (ab79559, Abcam BD Biosciences), CD63 (ab134045, Santa Cruz Biotechnology), TSG101 (ab125011, GAPDH (2118, Cell Signaling Technology) and Santa Cruz Biotechnology).

    Techniques:

    E2F2 is the target gene of miR-631, and elevated E2F2 facilitates the malignant behavior of NSCLC.

    Journal: Bioengineered

    Article Title: MicroRNA-631 deriving from bone marrow mesenchymal stem cell exosomes facilitates the malignant behavior of non-small cell lung cancer via modulating the E2F family of transcription factor 2/phosphatidylinositol 3‐kinase/Akt signaling pathway

    doi: 10.1080/21655979.2022.2036891

    Figure Lengend Snippet: E2F2 is the target gene of miR-631, and elevated E2F2 facilitates the malignant behavior of NSCLC.

    Article Snippet: A total of 20 g samples were applied to 12% sulfate-polyacrylamide gel, electrophoresis was performed to separate proteins, and then electroblot was onto polyvinylidene fluoride membrane (Invitrogen, Carlsbad, CA, USA), which was sealed with 5% skimmed milk powder for 1 h. Subsequently, incubation of the membrane was with the following primary antibodies: CD9 (ab92726, Abcam), E2F2 (14,129, 1:1000, Cell Signaling Technology), CD81 (ab79559, Abcam BD Biosciences), CD63 (ab134045, Santa Cruz Biotechnology), TSG101 (ab125011, GAPDH (2118, Cell Signaling Technology) and Santa Cruz Biotechnology).

    Techniques:

    MiR-631 exerts an influence on the malignant behavior of NSCLC via modulating E2F2.

    Journal: Bioengineered

    Article Title: MicroRNA-631 deriving from bone marrow mesenchymal stem cell exosomes facilitates the malignant behavior of non-small cell lung cancer via modulating the E2F family of transcription factor 2/phosphatidylinositol 3‐kinase/Akt signaling pathway

    doi: 10.1080/21655979.2022.2036891

    Figure Lengend Snippet: MiR-631 exerts an influence on the malignant behavior of NSCLC via modulating E2F2.

    Article Snippet: A total of 20 g samples were applied to 12% sulfate-polyacrylamide gel, electrophoresis was performed to separate proteins, and then electroblot was onto polyvinylidene fluoride membrane (Invitrogen, Carlsbad, CA, USA), which was sealed with 5% skimmed milk powder for 1 h. Subsequently, incubation of the membrane was with the following primary antibodies: CD9 (ab92726, Abcam), E2F2 (14,129, 1:1000, Cell Signaling Technology), CD81 (ab79559, Abcam BD Biosciences), CD63 (ab134045, Santa Cruz Biotechnology), TSG101 (ab125011, GAPDH (2118, Cell Signaling Technology) and Santa Cruz Biotechnology).

    Techniques:

    BMSCs-Exo exerts an influence on the malignant behavior of NSCLC via the miR-631/E2F2 axis.

    Journal: Bioengineered

    Article Title: MicroRNA-631 deriving from bone marrow mesenchymal stem cell exosomes facilitates the malignant behavior of non-small cell lung cancer via modulating the E2F family of transcription factor 2/phosphatidylinositol 3‐kinase/Akt signaling pathway

    doi: 10.1080/21655979.2022.2036891

    Figure Lengend Snippet: BMSCs-Exo exerts an influence on the malignant behavior of NSCLC via the miR-631/E2F2 axis.

    Article Snippet: A total of 20 g samples were applied to 12% sulfate-polyacrylamide gel, electrophoresis was performed to separate proteins, and then electroblot was onto polyvinylidene fluoride membrane (Invitrogen, Carlsbad, CA, USA), which was sealed with 5% skimmed milk powder for 1 h. Subsequently, incubation of the membrane was with the following primary antibodies: CD9 (ab92726, Abcam), E2F2 (14,129, 1:1000, Cell Signaling Technology), CD81 (ab79559, Abcam BD Biosciences), CD63 (ab134045, Santa Cruz Biotechnology), TSG101 (ab125011, GAPDH (2118, Cell Signaling Technology) and Santa Cruz Biotechnology).

    Techniques:

    a Violin plots showing the average expression levels of components of PRC2 in the Mix, Proxi, and Dista clusters. To increase the reproducibility, the gene expressions of H3K27me3 and H3K4me3 scSET-seq were merged for plotting. P values were calculated by Student’s t -test, two-sided. Rbbp7 was not detected in the scSET-seq. n = 291 (Mix), 133 (Proxi), and 121 (Dista) cells. b Western blot showing the indicated protein levels in parental and KO mESCs. Ezh2 KO decreased the total levels of H3K27me3 and increased the H3K27ac. Aebp2 KO didn’t exhibit obvious effects on the total levels of tested histone marks. Cell extracts were analyzed via Western blotting using the indicated antibodies, and two biological replicates were performed for each blot. Star indicated a nonspecific band. See Supplementary Fig. for the Sanger sequencing results of KO clones. c Representative images of divided Nanog - mCherry mESCs cocultured with Wnt3a beads. Wnt3a beads and cells were marked in the immunofluorescence image. Asymmetric, higher mCherry amounts in the bead-proximal cell; Symmetric, similar amounts of mCherry in either cell; Reverse, higher amounts of mCherry in the bead-distal cell. BF, bright field. Scale bar, 15 µm. d The percentages of asymmetrically, symmetrically, and reversely divided cells, as defined by Nanog - mCherry signals, in parental, Ezh2 and Aebp2 KO cells. P values were calculated by chi-squared test. e The enrichments of AEBP2 at cluster marker genes that were identified by gene expression. AEBP2 peaks were first called and annotated to the closet transcription starting sites within 20 Kb. The reads density of Aebp2 was then calculated for peaks annotated to each gene. P values were calculated by Student’s t -test, two-sided. The boxes were drawn from lower quartile (Q1) to upper quartile (Q3) with the middle line denoting the median, and whiskers with maximum 1.5 IQR (interquartile range). n = 90, 108, and 200 Aebp2 peaks at Mix, Proxi and Dista markers, respectively. n = 1000 (Mix), 1000 (Proxi), and 1000 (Dista) bins of Aebp2 at cluster marker genes. f The enrichments of H3K27me3 at cluster marker genes that were identified by gene expression. H3K27me3 peaks were first called in WT and Aebp2 KO cells, merged, and annotated to the closet transcription starting sites within 20 Kb. The reads density of H3K27me3 was then calculated for peaks annotated to each gene. P values were calculated by Student’s t-test, two-sided. The boxes were drawn from lower quartile (Q1) to upper quartile (Q3) with the middle line denoting the median, and whiskers with maximum 1.5 IQR (interquartile range). n = 504, 243, and 371 H3K27me3 peaks at Mix, Proxi, and Dista markers, respectively. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Joint single-cell multiomic analysis in Wnt3a induced asymmetric stem cell division

    doi: 10.1038/s41467-021-26203-0

    Figure Lengend Snippet: a Violin plots showing the average expression levels of components of PRC2 in the Mix, Proxi, and Dista clusters. To increase the reproducibility, the gene expressions of H3K27me3 and H3K4me3 scSET-seq were merged for plotting. P values were calculated by Student’s t -test, two-sided. Rbbp7 was not detected in the scSET-seq. n = 291 (Mix), 133 (Proxi), and 121 (Dista) cells. b Western blot showing the indicated protein levels in parental and KO mESCs. Ezh2 KO decreased the total levels of H3K27me3 and increased the H3K27ac. Aebp2 KO didn’t exhibit obvious effects on the total levels of tested histone marks. Cell extracts were analyzed via Western blotting using the indicated antibodies, and two biological replicates were performed for each blot. Star indicated a nonspecific band. See Supplementary Fig. for the Sanger sequencing results of KO clones. c Representative images of divided Nanog - mCherry mESCs cocultured with Wnt3a beads. Wnt3a beads and cells were marked in the immunofluorescence image. Asymmetric, higher mCherry amounts in the bead-proximal cell; Symmetric, similar amounts of mCherry in either cell; Reverse, higher amounts of mCherry in the bead-distal cell. BF, bright field. Scale bar, 15 µm. d The percentages of asymmetrically, symmetrically, and reversely divided cells, as defined by Nanog - mCherry signals, in parental, Ezh2 and Aebp2 KO cells. P values were calculated by chi-squared test. e The enrichments of AEBP2 at cluster marker genes that were identified by gene expression. AEBP2 peaks were first called and annotated to the closet transcription starting sites within 20 Kb. The reads density of Aebp2 was then calculated for peaks annotated to each gene. P values were calculated by Student’s t -test, two-sided. The boxes were drawn from lower quartile (Q1) to upper quartile (Q3) with the middle line denoting the median, and whiskers with maximum 1.5 IQR (interquartile range). n = 90, 108, and 200 Aebp2 peaks at Mix, Proxi and Dista markers, respectively. n = 1000 (Mix), 1000 (Proxi), and 1000 (Dista) bins of Aebp2 at cluster marker genes. f The enrichments of H3K27me3 at cluster marker genes that were identified by gene expression. H3K27me3 peaks were first called in WT and Aebp2 KO cells, merged, and annotated to the closet transcription starting sites within 20 Kb. The reads density of H3K27me3 was then calculated for peaks annotated to each gene. P values were calculated by Student’s t-test, two-sided. The boxes were drawn from lower quartile (Q1) to upper quartile (Q3) with the middle line denoting the median, and whiskers with maximum 1.5 IQR (interquartile range). n = 504, 243, and 371 H3K27me3 peaks at Mix, Proxi, and Dista markers, respectively. Source data are provided as a Source Data file.

    Article Snippet: Rabbit polyclonal anti-Histone H3 (Cat.# ab1791, Abcam), 1:5000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K27me3 (Cat.# 9733, Cell Signaling Technology), 1:1000 diluted for Western blot, 1:50 diluted for SET-seq, 1:200 diluted for scSET-seq; Rabbit polyclonal anti-Histone H3K4me3 (Cat.# ab8580, Abcam), 1:1000 diluted for Western blot, 1:50 diluted for SET-seq, 1:200 diluted for scSET-seq; Rabbit polyclonal anti-Histone H3K9me3 (Cat.# ab8898, Abcam), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K27ac (Cat.# ab4729, Abcam), 1:1000 diluted for Western blot; Rabbit monoclonal anti-EZH2 (Cat.# 5246, Clone name D2C9, Cell Signaling Technology), 1:1000 diluted for Western blot; Rabbit monoclonal anti-AEBP2 (Cat.# 14129, Clone name D7C6X, Cell Signaling Technology), 1:1000 diluted for Western blot; Rabbit polyclonal anti-JARID2 (Cat.# G-2, Novus Biologicals), 1:1000 diluted for Western blot; Peroxidase AffiniPure Goat anti-Rabbit IgG (H + L) (Cat.# 111-035-003, Jackson ImmunoResearch Laboratories), 1:1000 diluted for Western blot.

    Techniques: Expressing, Western Blot, Sequencing, Clone Assay, Immunofluorescence, Marker

    PRC2 sub-complexes differentially contribute to the BAP1-dependent intergenic H3K27me3 deposition (A) Cartoon showing divergent compositions of the PRC2.1 and PRC2.2 complexes and their differing affinities for chromatin features. (B) Western blot analysis with the indicated antibodies on total protein extracts from the Bap1 KO, Aebp2 + Jarid2 ( A+J ) KO, Aebp2 + Jarid2 + Bap1 ( A+J+B ) KO, and matching WT ESCs. (C) Western blot analysis with the indicated antibodies on total protein extracts from the indicated ESC lines. (D) Boxplots representing H2AK119ub1 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (E) Boxplots representing H3K27me3 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (F) Boxplots showing the log2 fold change RPKM ratio for H2AK119ub1 or H3K27me3 in the indicated cell lines (A = Aebp2 , J = Jarid2 , P = Pcl1-3 ) at intergenic regions (n = 38,068). (G) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Aebp2/Jarid2/Bap1 KO versus Aebp2/Jarid2 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. (H) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Pcl1-3/Bap1 KO versus Pcl1-3 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. See also <xref ref-type=Figure S4 . " width="100%" height="100%">

    Journal: Molecular Cell

    Article Title: BAP1 enhances Polycomb repression by counteracting widespread H2AK119ub1 deposition and chromatin condensation

    doi: 10.1016/j.molcel.2021.06.020

    Figure Lengend Snippet: PRC2 sub-complexes differentially contribute to the BAP1-dependent intergenic H3K27me3 deposition (A) Cartoon showing divergent compositions of the PRC2.1 and PRC2.2 complexes and their differing affinities for chromatin features. (B) Western blot analysis with the indicated antibodies on total protein extracts from the Bap1 KO, Aebp2 + Jarid2 ( A+J ) KO, Aebp2 + Jarid2 + Bap1 ( A+J+B ) KO, and matching WT ESCs. (C) Western blot analysis with the indicated antibodies on total protein extracts from the indicated ESC lines. (D) Boxplots representing H2AK119ub1 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (E) Boxplots representing H3K27me3 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (F) Boxplots showing the log2 fold change RPKM ratio for H2AK119ub1 or H3K27me3 in the indicated cell lines (A = Aebp2 , J = Jarid2 , P = Pcl1-3 ) at intergenic regions (n = 38,068). (G) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Aebp2/Jarid2/Bap1 KO versus Aebp2/Jarid2 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. (H) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Pcl1-3/Bap1 KO versus Pcl1-3 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. See also Figure S4 .

    Article Snippet: Rabbit monoclonal anti-AEBP2 (D7C6X) , Cell Signaling , Cat #14129; RRID: AB_2798398.

    Techniques: Western Blot, ChIP-sequencing, Genome Wide

    Journal: Molecular Cell

    Article Title: BAP1 enhances Polycomb repression by counteracting widespread H2AK119ub1 deposition and chromatin condensation

    doi: 10.1016/j.molcel.2021.06.020

    Figure Lengend Snippet:

    Article Snippet: Rabbit monoclonal anti-AEBP2 (D7C6X) , Cell Signaling , Cat #14129; RRID: AB_2798398.

    Techniques: Recombinant, Transfection, Purification, Sequencing, Imaging, Plasmid Preparation, Software