anti h2b 2934s  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti h2b 2934s
    A Representative image for the dynamic recruitment of WT or 3A GFP-RAD18 to laser-induced DSBs. Data were presented as mean ± SEM from 15 cells. Scale bar, 10 μm. B Quantification of the time course of WT or 3A GFP-RAD18 recruitment after laser microirradiation. C Representative images of GFP-RAD18 foci stained with DAPI after UV irradiation. Scale bars: 2 μm. The protein levels of RAD18 in RAD18+/+ and RAD18-/- U2OS cells were detected by immunoblotting. D Quantification of the percentage of RAD18-/- U2OS cells transfected with WT or 3A GFP-RAD18 constructs with more than 30 RAD18 foci after CPT, Bleomycin and UV exposure by counting at least 200 cells in each experiment. Data represent means ± SEM from three independent experiments. E Chromatin fractions of HEK293T cells expressing WT or 3A SFB-RAD18 were extracted followed by immunoblotting with Flag and <t>H2B</t> antibodies. The whole cell extract (WCE) was harvested and immunoblotted with Flag and β-actin antibodies.
    Anti H2b 2934s, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "RAD18 O-GlcNAcylation promotes translesion DNA synthesis and homologous recombination repair"

    Article Title: RAD18 O-GlcNAcylation promotes translesion DNA synthesis and homologous recombination repair

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-024-06700-y

    A Representative image for the dynamic recruitment of WT or 3A GFP-RAD18 to laser-induced DSBs. Data were presented as mean ± SEM from 15 cells. Scale bar, 10 μm. B Quantification of the time course of WT or 3A GFP-RAD18 recruitment after laser microirradiation. C Representative images of GFP-RAD18 foci stained with DAPI after UV irradiation. Scale bars: 2 μm. The protein levels of RAD18 in RAD18+/+ and RAD18-/- U2OS cells were detected by immunoblotting. D Quantification of the percentage of RAD18-/- U2OS cells transfected with WT or 3A GFP-RAD18 constructs with more than 30 RAD18 foci after CPT, Bleomycin and UV exposure by counting at least 200 cells in each experiment. Data represent means ± SEM from three independent experiments. E Chromatin fractions of HEK293T cells expressing WT or 3A SFB-RAD18 were extracted followed by immunoblotting with Flag and H2B antibodies. The whole cell extract (WCE) was harvested and immunoblotted with Flag and β-actin antibodies.
    Figure Legend Snippet: A Representative image for the dynamic recruitment of WT or 3A GFP-RAD18 to laser-induced DSBs. Data were presented as mean ± SEM from 15 cells. Scale bar, 10 μm. B Quantification of the time course of WT or 3A GFP-RAD18 recruitment after laser microirradiation. C Representative images of GFP-RAD18 foci stained with DAPI after UV irradiation. Scale bars: 2 μm. The protein levels of RAD18 in RAD18+/+ and RAD18-/- U2OS cells were detected by immunoblotting. D Quantification of the percentage of RAD18-/- U2OS cells transfected with WT or 3A GFP-RAD18 constructs with more than 30 RAD18 foci after CPT, Bleomycin and UV exposure by counting at least 200 cells in each experiment. Data represent means ± SEM from three independent experiments. E Chromatin fractions of HEK293T cells expressing WT or 3A SFB-RAD18 were extracted followed by immunoblotting with Flag and H2B antibodies. The whole cell extract (WCE) was harvested and immunoblotted with Flag and β-actin antibodies.

    Techniques Used: Staining, Irradiation, Western Blot, Transfection, Construct, Expressing

    histone h2b  (Cell Signaling Technology Inc)


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    anti h2b  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti h2b
    (A) RQ-PCR analysis of MLL in SU-DHL-5 (expression level was set to unity) and in leukemia/lymphoma control cell lines. (B) RQ-PCR analysis of MLL, NKX2-1 and HEY1 in siRNA-treated SU-DHL-5 cells (left). ChIP analysis of the NKX2-1 and HEY1 promoters in SU-DHL-5 and SU-DHL-4 (for control) showed representation of particular histone H3 modifications, as shown by PCR amplification of genomic fragments (right). Untreated genomic DNA served as positive control, NTC: no template control. (C) Copy number analysis by genomic profiling indicates presence of aberrations at MLL at 11q23. Inserts show an enlargement of chromosome 11 obtained by SKY karyotyping and an enlargement of the MLL locus obtained by genomic profiling. (D) FISH analysis of the MLL locus in SU-DHL-5 (below) using BAC probes as indicated above. The results show one wild type allele and one amplified MLL locus. (E) RT-PCR analysis of MLL fusion genes in SU-DHL-5 and particular positive and negative control cell lines. TEL expression served as control, NTC: no template control. (F) Copy number analysis by genomic profiling of chromosome 6 indicates extended deletions at both arms. The HIST1 locus maps to the breakpoint region at 6p22. Genes located in deleted regions include JARID2, SOX4, HMGN3, AKAP7 and MAP3K4. Insert shows chromosomes 6 analyzed by SKY karyotyping indicating rearrangements at both chromosomes. (G) FISH analysis of the histone gene cluster HIST1 at 6p22 (below) using painting probe and BACs as indicated above. (H) RQ-PCR analysis of selected histone genes in SU-DHL-5 and SU-DHL-4 for control (left). PAGE analysis of histone proteins in three DLBCL cell lines (middle) demonstrates elevated levels in SU-DHL-5. Western blot analysis of <t>H2B,</t> H2Bub1 and ERK (for control) in four DLBCL cell lines (right) demonstrates elevated levels in SU-DHL-5.
    Anti H2b, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Ectopic Expression of Homeobox Gene NKX2-1 in Diffuse Large B-Cell Lymphoma Is Mediated by Aberrant Chromatin Modifications"

    Article Title: Ectopic Expression of Homeobox Gene NKX2-1 in Diffuse Large B-Cell Lymphoma Is Mediated by Aberrant Chromatin Modifications

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061447

    (A) RQ-PCR analysis of MLL in SU-DHL-5 (expression level was set to unity) and in leukemia/lymphoma control cell lines. (B) RQ-PCR analysis of MLL, NKX2-1 and HEY1 in siRNA-treated SU-DHL-5 cells (left). ChIP analysis of the NKX2-1 and HEY1 promoters in SU-DHL-5 and SU-DHL-4 (for control) showed representation of particular histone H3 modifications, as shown by PCR amplification of genomic fragments (right). Untreated genomic DNA served as positive control, NTC: no template control. (C) Copy number analysis by genomic profiling indicates presence of aberrations at MLL at 11q23. Inserts show an enlargement of chromosome 11 obtained by SKY karyotyping and an enlargement of the MLL locus obtained by genomic profiling. (D) FISH analysis of the MLL locus in SU-DHL-5 (below) using BAC probes as indicated above. The results show one wild type allele and one amplified MLL locus. (E) RT-PCR analysis of MLL fusion genes in SU-DHL-5 and particular positive and negative control cell lines. TEL expression served as control, NTC: no template control. (F) Copy number analysis by genomic profiling of chromosome 6 indicates extended deletions at both arms. The HIST1 locus maps to the breakpoint region at 6p22. Genes located in deleted regions include JARID2, SOX4, HMGN3, AKAP7 and MAP3K4. Insert shows chromosomes 6 analyzed by SKY karyotyping indicating rearrangements at both chromosomes. (G) FISH analysis of the histone gene cluster HIST1 at 6p22 (below) using painting probe and BACs as indicated above. (H) RQ-PCR analysis of selected histone genes in SU-DHL-5 and SU-DHL-4 for control (left). PAGE analysis of histone proteins in three DLBCL cell lines (middle) demonstrates elevated levels in SU-DHL-5. Western blot analysis of H2B, H2Bub1 and ERK (for control) in four DLBCL cell lines (right) demonstrates elevated levels in SU-DHL-5.
    Figure Legend Snippet: (A) RQ-PCR analysis of MLL in SU-DHL-5 (expression level was set to unity) and in leukemia/lymphoma control cell lines. (B) RQ-PCR analysis of MLL, NKX2-1 and HEY1 in siRNA-treated SU-DHL-5 cells (left). ChIP analysis of the NKX2-1 and HEY1 promoters in SU-DHL-5 and SU-DHL-4 (for control) showed representation of particular histone H3 modifications, as shown by PCR amplification of genomic fragments (right). Untreated genomic DNA served as positive control, NTC: no template control. (C) Copy number analysis by genomic profiling indicates presence of aberrations at MLL at 11q23. Inserts show an enlargement of chromosome 11 obtained by SKY karyotyping and an enlargement of the MLL locus obtained by genomic profiling. (D) FISH analysis of the MLL locus in SU-DHL-5 (below) using BAC probes as indicated above. The results show one wild type allele and one amplified MLL locus. (E) RT-PCR analysis of MLL fusion genes in SU-DHL-5 and particular positive and negative control cell lines. TEL expression served as control, NTC: no template control. (F) Copy number analysis by genomic profiling of chromosome 6 indicates extended deletions at both arms. The HIST1 locus maps to the breakpoint region at 6p22. Genes located in deleted regions include JARID2, SOX4, HMGN3, AKAP7 and MAP3K4. Insert shows chromosomes 6 analyzed by SKY karyotyping indicating rearrangements at both chromosomes. (G) FISH analysis of the histone gene cluster HIST1 at 6p22 (below) using painting probe and BACs as indicated above. (H) RQ-PCR analysis of selected histone genes in SU-DHL-5 and SU-DHL-4 for control (left). PAGE analysis of histone proteins in three DLBCL cell lines (middle) demonstrates elevated levels in SU-DHL-5. Western blot analysis of H2B, H2Bub1 and ERK (for control) in four DLBCL cell lines (right) demonstrates elevated levels in SU-DHL-5.

    Techniques Used: Expressing, Amplification, Positive Control, Reverse Transcription Polymerase Chain Reaction, Negative Control, Western Blot

    The figure summarizes the regulations of the genes described in this study, highlighting a central position of NKX2-1. Chromosomal aberrations activate expression of MLL (11q23) and histones including H2B (6p22). MLL together with H2Bub1 generate an activatory chromatin structure at NKX2-1. This structure is reinforced by reduced expression of USP46 and E2F6 and elevated expression of RNF20/40, JMJD3 and HOPX, and mediates activation of NKX2-1. NKX2-1 in turn activates directly expression of HEY1 which performs inhibition of B-cell differentiation. Both, NKX2-1 and HEY1 contribute to the activatory chromatin structure by regulating RNF40 and E2F6, respectively. IL4/STAT3-signaling enhances expression of NKX2-1. TNFa, cGMP, cAMP and PRKCE support NFkB which activates both NKX2-1 and HEY1. Reduced expression of PDE6D and enhanced expression of NOS1 contribute to elevated cGMP. NOS1 and PRKCE are activated by NKX2-1. HEY1 mediates reduced expression of PDE4A resulting in elevated cAMP levels. Finally, NOTCH-signaling and TGFb-signaling (via SMAD) activate HEY1 expression. SMAD and NKX2-1 interact and coactivate HEY1 transcription.
    Figure Legend Snippet: The figure summarizes the regulations of the genes described in this study, highlighting a central position of NKX2-1. Chromosomal aberrations activate expression of MLL (11q23) and histones including H2B (6p22). MLL together with H2Bub1 generate an activatory chromatin structure at NKX2-1. This structure is reinforced by reduced expression of USP46 and E2F6 and elevated expression of RNF20/40, JMJD3 and HOPX, and mediates activation of NKX2-1. NKX2-1 in turn activates directly expression of HEY1 which performs inhibition of B-cell differentiation. Both, NKX2-1 and HEY1 contribute to the activatory chromatin structure by regulating RNF40 and E2F6, respectively. IL4/STAT3-signaling enhances expression of NKX2-1. TNFa, cGMP, cAMP and PRKCE support NFkB which activates both NKX2-1 and HEY1. Reduced expression of PDE6D and enhanced expression of NOS1 contribute to elevated cGMP. NOS1 and PRKCE are activated by NKX2-1. HEY1 mediates reduced expression of PDE4A resulting in elevated cAMP levels. Finally, NOTCH-signaling and TGFb-signaling (via SMAD) activate HEY1 expression. SMAD and NKX2-1 interact and coactivate HEY1 transcription.

    Techniques Used: Expressing, Activation Assay, Inhibition, Cell Differentiation

    anti h2b  (Cell Signaling Technology Inc)


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    histone h2b  (Cell Signaling Technology Inc)


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    histone h2b  (Cell Signaling Technology Inc)


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    anti h2b monoclonal antibody  (Cell Signaling Technology Inc)


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    (A, B, C, D) Close-up views of the SET8 Arg188 and Arg192 residues bound to the H2A Glu56 and <t>H2B</t> Glu113 residues (B, D, left) and the H2A Glu61 and Glu92 residues (B, D, right), respectively, in the acidic patches of the NCP (A, B) and the NCP CENP-A (C, D). The atomic model of SET8 (PDB: 1ZKK ) was docked into the EM density maps of the SET8–NCP and SET8–NCP CENP-A complexes.
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    1) Product Images from "Structural basis of nucleosomal histone H4 lysine 20 methylation by SET8 methyltransferase"

    Article Title: Structural basis of nucleosomal histone H4 lysine 20 methylation by SET8 methyltransferase

    Journal: Life Science Alliance

    doi: 10.26508/lsa.202000919

    (A, B, C, D) Close-up views of the SET8 Arg188 and Arg192 residues bound to the H2A Glu56 and H2B Glu113 residues (B, D, left) and the H2A Glu61 and Glu92 residues (B, D, right), respectively, in the acidic patches of the NCP (A, B) and the NCP CENP-A (C, D). The atomic model of SET8 (PDB: 1ZKK ) was docked into the EM density maps of the SET8–NCP and SET8–NCP CENP-A complexes.
    Figure Legend Snippet: (A, B, C, D) Close-up views of the SET8 Arg188 and Arg192 residues bound to the H2A Glu56 and H2B Glu113 residues (B, D, left) and the H2A Glu61 and Glu92 residues (B, D, right), respectively, in the acidic patches of the NCP (A, B) and the NCP CENP-A (C, D). The atomic model of SET8 (PDB: 1ZKK ) was docked into the EM density maps of the SET8–NCP and SET8–NCP CENP-A complexes.

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    Cell Signaling Technology Inc anti h2b 2934s
    A Representative image for the dynamic recruitment of WT or 3A GFP-RAD18 to laser-induced DSBs. Data were presented as mean ± SEM from 15 cells. Scale bar, 10 μm. B Quantification of the time course of WT or 3A GFP-RAD18 recruitment after laser microirradiation. C Representative images of GFP-RAD18 foci stained with DAPI after UV irradiation. Scale bars: 2 μm. The protein levels of RAD18 in RAD18+/+ and RAD18-/- U2OS cells were detected by immunoblotting. D Quantification of the percentage of RAD18-/- U2OS cells transfected with WT or 3A GFP-RAD18 constructs with more than 30 RAD18 foci after CPT, Bleomycin and UV exposure by counting at least 200 cells in each experiment. Data represent means ± SEM from three independent experiments. E Chromatin fractions of HEK293T cells expressing WT or 3A SFB-RAD18 were extracted followed by immunoblotting with Flag and <t>H2B</t> antibodies. The whole cell extract (WCE) was harvested and immunoblotted with Flag and β-actin antibodies.
    Anti H2b 2934s, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A Representative image for the dynamic recruitment of WT or 3A GFP-RAD18 to laser-induced DSBs. Data were presented as mean ± SEM from 15 cells. Scale bar, 10 μm. B Quantification of the time course of WT or 3A GFP-RAD18 recruitment after laser microirradiation. C Representative images of GFP-RAD18 foci stained with DAPI after UV irradiation. Scale bars: 2 μm. The protein levels of RAD18 in RAD18+/+ and RAD18-/- U2OS cells were detected by immunoblotting. D Quantification of the percentage of RAD18-/- U2OS cells transfected with WT or 3A GFP-RAD18 constructs with more than 30 RAD18 foci after CPT, Bleomycin and UV exposure by counting at least 200 cells in each experiment. Data represent means ± SEM from three independent experiments. E Chromatin fractions of HEK293T cells expressing WT or 3A SFB-RAD18 were extracted followed by immunoblotting with Flag and <t>H2B</t> antibodies. The whole cell extract (WCE) was harvested and immunoblotted with Flag and β-actin antibodies.
    Histone H2b, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) RQ-PCR analysis of MLL in SU-DHL-5 (expression level was set to unity) and in leukemia/lymphoma control cell lines. (B) RQ-PCR analysis of MLL, NKX2-1 and HEY1 in siRNA-treated SU-DHL-5 cells (left). ChIP analysis of the NKX2-1 and HEY1 promoters in SU-DHL-5 and SU-DHL-4 (for control) showed representation of particular histone H3 modifications, as shown by PCR amplification of genomic fragments (right). Untreated genomic DNA served as positive control, NTC: no template control. (C) Copy number analysis by genomic profiling indicates presence of aberrations at MLL at 11q23. Inserts show an enlargement of chromosome 11 obtained by SKY karyotyping and an enlargement of the MLL locus obtained by genomic profiling. (D) FISH analysis of the MLL locus in SU-DHL-5 (below) using BAC probes as indicated above. The results show one wild type allele and one amplified MLL locus. (E) RT-PCR analysis of MLL fusion genes in SU-DHL-5 and particular positive and negative control cell lines. TEL expression served as control, NTC: no template control. (F) Copy number analysis by genomic profiling of chromosome 6 indicates extended deletions at both arms. The HIST1 locus maps to the breakpoint region at 6p22. Genes located in deleted regions include JARID2, SOX4, HMGN3, AKAP7 and MAP3K4. Insert shows chromosomes 6 analyzed by SKY karyotyping indicating rearrangements at both chromosomes. (G) FISH analysis of the histone gene cluster HIST1 at 6p22 (below) using painting probe and BACs as indicated above. (H) RQ-PCR analysis of selected histone genes in SU-DHL-5 and SU-DHL-4 for control (left). PAGE analysis of histone proteins in three DLBCL cell lines (middle) demonstrates elevated levels in SU-DHL-5. Western blot analysis of <t>H2B,</t> H2Bub1 and ERK (for control) in four DLBCL cell lines (right) demonstrates elevated levels in SU-DHL-5.
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    (A) RQ-PCR analysis of MLL in SU-DHL-5 (expression level was set to unity) and in leukemia/lymphoma control cell lines. (B) RQ-PCR analysis of MLL, NKX2-1 and HEY1 in siRNA-treated SU-DHL-5 cells (left). ChIP analysis of the NKX2-1 and HEY1 promoters in SU-DHL-5 and SU-DHL-4 (for control) showed representation of particular histone H3 modifications, as shown by PCR amplification of genomic fragments (right). Untreated genomic DNA served as positive control, NTC: no template control. (C) Copy number analysis by genomic profiling indicates presence of aberrations at MLL at 11q23. Inserts show an enlargement of chromosome 11 obtained by SKY karyotyping and an enlargement of the MLL locus obtained by genomic profiling. (D) FISH analysis of the MLL locus in SU-DHL-5 (below) using BAC probes as indicated above. The results show one wild type allele and one amplified MLL locus. (E) RT-PCR analysis of MLL fusion genes in SU-DHL-5 and particular positive and negative control cell lines. TEL expression served as control, NTC: no template control. (F) Copy number analysis by genomic profiling of chromosome 6 indicates extended deletions at both arms. The HIST1 locus maps to the breakpoint region at 6p22. Genes located in deleted regions include JARID2, SOX4, HMGN3, AKAP7 and MAP3K4. Insert shows chromosomes 6 analyzed by SKY karyotyping indicating rearrangements at both chromosomes. (G) FISH analysis of the histone gene cluster HIST1 at 6p22 (below) using painting probe and BACs as indicated above. (H) RQ-PCR analysis of selected histone genes in SU-DHL-5 and SU-DHL-4 for control (left). PAGE analysis of histone proteins in three DLBCL cell lines (middle) demonstrates elevated levels in SU-DHL-5. Western blot analysis of <t>H2B,</t> H2Bub1 and ERK (for control) in four DLBCL cell lines (right) demonstrates elevated levels in SU-DHL-5.
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    Cell Signaling Technology Inc anti h2b monoclonal antibody
    (A, B, C, D) Close-up views of the SET8 Arg188 and Arg192 residues bound to the H2A Glu56 and <t>H2B</t> Glu113 residues (B, D, left) and the H2A Glu61 and Glu92 residues (B, D, right), respectively, in the acidic patches of the NCP (A, B) and the NCP CENP-A (C, D). The atomic model of SET8 (PDB: 1ZKK ) was docked into the EM density maps of the SET8–NCP and SET8–NCP CENP-A complexes.
    Anti H2b Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A Representative image for the dynamic recruitment of WT or 3A GFP-RAD18 to laser-induced DSBs. Data were presented as mean ± SEM from 15 cells. Scale bar, 10 μm. B Quantification of the time course of WT or 3A GFP-RAD18 recruitment after laser microirradiation. C Representative images of GFP-RAD18 foci stained with DAPI after UV irradiation. Scale bars: 2 μm. The protein levels of RAD18 in RAD18+/+ and RAD18-/- U2OS cells were detected by immunoblotting. D Quantification of the percentage of RAD18-/- U2OS cells transfected with WT or 3A GFP-RAD18 constructs with more than 30 RAD18 foci after CPT, Bleomycin and UV exposure by counting at least 200 cells in each experiment. Data represent means ± SEM from three independent experiments. E Chromatin fractions of HEK293T cells expressing WT or 3A SFB-RAD18 were extracted followed by immunoblotting with Flag and H2B antibodies. The whole cell extract (WCE) was harvested and immunoblotted with Flag and β-actin antibodies.

    Journal: Cell Death & Disease

    Article Title: RAD18 O-GlcNAcylation promotes translesion DNA synthesis and homologous recombination repair

    doi: 10.1038/s41419-024-06700-y

    Figure Lengend Snippet: A Representative image for the dynamic recruitment of WT or 3A GFP-RAD18 to laser-induced DSBs. Data were presented as mean ± SEM from 15 cells. Scale bar, 10 μm. B Quantification of the time course of WT or 3A GFP-RAD18 recruitment after laser microirradiation. C Representative images of GFP-RAD18 foci stained with DAPI after UV irradiation. Scale bars: 2 μm. The protein levels of RAD18 in RAD18+/+ and RAD18-/- U2OS cells were detected by immunoblotting. D Quantification of the percentage of RAD18-/- U2OS cells transfected with WT or 3A GFP-RAD18 constructs with more than 30 RAD18 foci after CPT, Bleomycin and UV exposure by counting at least 200 cells in each experiment. Data represent means ± SEM from three independent experiments. E Chromatin fractions of HEK293T cells expressing WT or 3A SFB-RAD18 were extracted followed by immunoblotting with Flag and H2B antibodies. The whole cell extract (WCE) was harvested and immunoblotted with Flag and β-actin antibodies.

    Article Snippet: Antibodies sources were as follows: mouse anti-Flag (F1804, 1:1000) from Sigma (St Louis, MO), anti-RAD18 (ab17725, 1:1000) from Abcam and (H00056852-M01) from Novus Biologicals, anti-RAD18 pS434 from Dia-An Biotechnology, O-linked β-N-acetylglucosamine (O-GlcNAc, ab2739, 1:2000) and anti-RAD51 (ab133534, 1:200) from Abcam, anti-HA (902302, 1:2000) from BioLegend, anti-Myc (MMS-150R-500, 1:1000) from Covance, anti-H3.1 (P30266, 1:2000) from Abmart, anti-β-Tubulin (AbM59005-37-PU, 1:4000) from Beijing Protein Innovation (Beijing, China), anti-GFP (sc-8334, 1:500), OGT (sc-32921, 1:1000) and anti-PCNA (sc-56, 1:1000) from Santa Cruz Biotechnology, anti-H2B (2934S) from Cell Signaling Technology (CST).

    Techniques: Staining, Irradiation, Western Blot, Transfection, Construct, Expressing

    (A) RQ-PCR analysis of MLL in SU-DHL-5 (expression level was set to unity) and in leukemia/lymphoma control cell lines. (B) RQ-PCR analysis of MLL, NKX2-1 and HEY1 in siRNA-treated SU-DHL-5 cells (left). ChIP analysis of the NKX2-1 and HEY1 promoters in SU-DHL-5 and SU-DHL-4 (for control) showed representation of particular histone H3 modifications, as shown by PCR amplification of genomic fragments (right). Untreated genomic DNA served as positive control, NTC: no template control. (C) Copy number analysis by genomic profiling indicates presence of aberrations at MLL at 11q23. Inserts show an enlargement of chromosome 11 obtained by SKY karyotyping and an enlargement of the MLL locus obtained by genomic profiling. (D) FISH analysis of the MLL locus in SU-DHL-5 (below) using BAC probes as indicated above. The results show one wild type allele and one amplified MLL locus. (E) RT-PCR analysis of MLL fusion genes in SU-DHL-5 and particular positive and negative control cell lines. TEL expression served as control, NTC: no template control. (F) Copy number analysis by genomic profiling of chromosome 6 indicates extended deletions at both arms. The HIST1 locus maps to the breakpoint region at 6p22. Genes located in deleted regions include JARID2, SOX4, HMGN3, AKAP7 and MAP3K4. Insert shows chromosomes 6 analyzed by SKY karyotyping indicating rearrangements at both chromosomes. (G) FISH analysis of the histone gene cluster HIST1 at 6p22 (below) using painting probe and BACs as indicated above. (H) RQ-PCR analysis of selected histone genes in SU-DHL-5 and SU-DHL-4 for control (left). PAGE analysis of histone proteins in three DLBCL cell lines (middle) demonstrates elevated levels in SU-DHL-5. Western blot analysis of H2B, H2Bub1 and ERK (for control) in four DLBCL cell lines (right) demonstrates elevated levels in SU-DHL-5.

    Journal: PLoS ONE

    Article Title: Ectopic Expression of Homeobox Gene NKX2-1 in Diffuse Large B-Cell Lymphoma Is Mediated by Aberrant Chromatin Modifications

    doi: 10.1371/journal.pone.0061447

    Figure Lengend Snippet: (A) RQ-PCR analysis of MLL in SU-DHL-5 (expression level was set to unity) and in leukemia/lymphoma control cell lines. (B) RQ-PCR analysis of MLL, NKX2-1 and HEY1 in siRNA-treated SU-DHL-5 cells (left). ChIP analysis of the NKX2-1 and HEY1 promoters in SU-DHL-5 and SU-DHL-4 (for control) showed representation of particular histone H3 modifications, as shown by PCR amplification of genomic fragments (right). Untreated genomic DNA served as positive control, NTC: no template control. (C) Copy number analysis by genomic profiling indicates presence of aberrations at MLL at 11q23. Inserts show an enlargement of chromosome 11 obtained by SKY karyotyping and an enlargement of the MLL locus obtained by genomic profiling. (D) FISH analysis of the MLL locus in SU-DHL-5 (below) using BAC probes as indicated above. The results show one wild type allele and one amplified MLL locus. (E) RT-PCR analysis of MLL fusion genes in SU-DHL-5 and particular positive and negative control cell lines. TEL expression served as control, NTC: no template control. (F) Copy number analysis by genomic profiling of chromosome 6 indicates extended deletions at both arms. The HIST1 locus maps to the breakpoint region at 6p22. Genes located in deleted regions include JARID2, SOX4, HMGN3, AKAP7 and MAP3K4. Insert shows chromosomes 6 analyzed by SKY karyotyping indicating rearrangements at both chromosomes. (G) FISH analysis of the histone gene cluster HIST1 at 6p22 (below) using painting probe and BACs as indicated above. (H) RQ-PCR analysis of selected histone genes in SU-DHL-5 and SU-DHL-4 for control (left). PAGE analysis of histone proteins in three DLBCL cell lines (middle) demonstrates elevated levels in SU-DHL-5. Western blot analysis of H2B, H2Bub1 and ERK (for control) in four DLBCL cell lines (right) demonstrates elevated levels in SU-DHL-5.

    Article Snippet: ChIP analysis was performed with the ChIP Assay Kit (Millipore-Upstate, Schwalbach, Germany) as described by the manufacturer, isolating genomic DNA fragments generated by sonication, using antibodies anti-NKX2-1 (EP1584Y, Abgent), anti-H2B (53H3, Cell Signaling, Danvers, MA, USA), anti-H2Bub1 (D11, Cell Signaling), anti-H3K4me3 (mAbcam1012, Abcam, Cambridge, UK) and anti-H3K27me3 (mAbcam6002, Abcam).

    Techniques: Expressing, Amplification, Positive Control, Reverse Transcription Polymerase Chain Reaction, Negative Control, Western Blot

    The figure summarizes the regulations of the genes described in this study, highlighting a central position of NKX2-1. Chromosomal aberrations activate expression of MLL (11q23) and histones including H2B (6p22). MLL together with H2Bub1 generate an activatory chromatin structure at NKX2-1. This structure is reinforced by reduced expression of USP46 and E2F6 and elevated expression of RNF20/40, JMJD3 and HOPX, and mediates activation of NKX2-1. NKX2-1 in turn activates directly expression of HEY1 which performs inhibition of B-cell differentiation. Both, NKX2-1 and HEY1 contribute to the activatory chromatin structure by regulating RNF40 and E2F6, respectively. IL4/STAT3-signaling enhances expression of NKX2-1. TNFa, cGMP, cAMP and PRKCE support NFkB which activates both NKX2-1 and HEY1. Reduced expression of PDE6D and enhanced expression of NOS1 contribute to elevated cGMP. NOS1 and PRKCE are activated by NKX2-1. HEY1 mediates reduced expression of PDE4A resulting in elevated cAMP levels. Finally, NOTCH-signaling and TGFb-signaling (via SMAD) activate HEY1 expression. SMAD and NKX2-1 interact and coactivate HEY1 transcription.

    Journal: PLoS ONE

    Article Title: Ectopic Expression of Homeobox Gene NKX2-1 in Diffuse Large B-Cell Lymphoma Is Mediated by Aberrant Chromatin Modifications

    doi: 10.1371/journal.pone.0061447

    Figure Lengend Snippet: The figure summarizes the regulations of the genes described in this study, highlighting a central position of NKX2-1. Chromosomal aberrations activate expression of MLL (11q23) and histones including H2B (6p22). MLL together with H2Bub1 generate an activatory chromatin structure at NKX2-1. This structure is reinforced by reduced expression of USP46 and E2F6 and elevated expression of RNF20/40, JMJD3 and HOPX, and mediates activation of NKX2-1. NKX2-1 in turn activates directly expression of HEY1 which performs inhibition of B-cell differentiation. Both, NKX2-1 and HEY1 contribute to the activatory chromatin structure by regulating RNF40 and E2F6, respectively. IL4/STAT3-signaling enhances expression of NKX2-1. TNFa, cGMP, cAMP and PRKCE support NFkB which activates both NKX2-1 and HEY1. Reduced expression of PDE6D and enhanced expression of NOS1 contribute to elevated cGMP. NOS1 and PRKCE are activated by NKX2-1. HEY1 mediates reduced expression of PDE4A resulting in elevated cAMP levels. Finally, NOTCH-signaling and TGFb-signaling (via SMAD) activate HEY1 expression. SMAD and NKX2-1 interact and coactivate HEY1 transcription.

    Article Snippet: ChIP analysis was performed with the ChIP Assay Kit (Millipore-Upstate, Schwalbach, Germany) as described by the manufacturer, isolating genomic DNA fragments generated by sonication, using antibodies anti-NKX2-1 (EP1584Y, Abgent), anti-H2B (53H3, Cell Signaling, Danvers, MA, USA), anti-H2Bub1 (D11, Cell Signaling), anti-H3K4me3 (mAbcam1012, Abcam, Cambridge, UK) and anti-H3K27me3 (mAbcam6002, Abcam).

    Techniques: Expressing, Activation Assay, Inhibition, Cell Differentiation

    (A, B, C, D) Close-up views of the SET8 Arg188 and Arg192 residues bound to the H2A Glu56 and H2B Glu113 residues (B, D, left) and the H2A Glu61 and Glu92 residues (B, D, right), respectively, in the acidic patches of the NCP (A, B) and the NCP CENP-A (C, D). The atomic model of SET8 (PDB: 1ZKK ) was docked into the EM density maps of the SET8–NCP and SET8–NCP CENP-A complexes.

    Journal: Life Science Alliance

    Article Title: Structural basis of nucleosomal histone H4 lysine 20 methylation by SET8 methyltransferase

    doi: 10.26508/lsa.202000919

    Figure Lengend Snippet: (A, B, C, D) Close-up views of the SET8 Arg188 and Arg192 residues bound to the H2A Glu56 and H2B Glu113 residues (B, D, left) and the H2A Glu61 and Glu92 residues (B, D, right), respectively, in the acidic patches of the NCP (A, B) and the NCP CENP-A (C, D). The atomic model of SET8 (PDB: 1ZKK ) was docked into the EM density maps of the SET8–NCP and SET8–NCP CENP-A complexes.

    Article Snippet: The membrane was washed with PBS-T and incubated with the primary antibodies, the mouse monoclonal antibody against monomethylated H4K20 (CMA421; 32 ) and the anti-H2B monoclonal antibody (53H3: Cell Signaling), diluted with Can Get Signal solution 1 (TOYOBO) at 4°C overnight.

    Techniques: