h3k9ac Abcam Search Results


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  • 95
    Millipore h3k9ac
    H3k9ac, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 745 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abcam anti h3k9ac
    BATF3 Inhibits Histone Acetylation at the Foxp3 Locus under iTreg-Polarizing Conditions (A) Immunoblot analysis of phosphorylated Smad3 and Stat5 in the nucleus of naive WT or Batf3-Tg CD4 + T cells at indicated hours upon iTreg polarization. (B) Quantitation as well as statistical analysis of the Smad3 and Stat5 expression in western blot experiments. (C and D) FACS sorted naive CD4 + Foxp3 − T cells (C and D) from WT or Batf3 -Tg Foxp3 -EGFP reporter mice were cultured under the iTreg-polarizing condition (with TGF-β and IL-2) or nonpolarizing condition conditions (without TGF-β and IL-2) for 48 hr. Shown are ChIP analysis of H3Ac, H4Ac (C), <t>H3K9Ac,</t> and H3K27Ac (D) at Foxp3 locus.
    Anti H3k9ac, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 551 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam h3k9ac
    Repressive histone modifications following GATA-1 overexpression in AML-ELs. ChIP at the PU . 1 gene locus was carried out for the H3K9Me3, H3K27Me3, and <t>H3K9Ac</t> histone tail modifications in OCI-M2 (left) and K562 (right) cells. Grey bars: control cells, dark bars: 48hrs after GATA-1 transgene transfection. Data are relative to control antibody IPs (Y axis). T-test significance: p
    H3k9ac, supplied by Abcam, used in various techniques. Bioz Stars score: 98/100, based on 1001 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    Abcam h3k9ac s10ph
    Repressive histone modifications following GATA-1 overexpression in AML-ELs. ChIP at the PU . 1 gene locus was carried out for the H3K9Me3, H3K27Me3, and <t>H3K9Ac</t> histone tail modifications in OCI-M2 (left) and K562 (right) cells. Grey bars: control cells, dark bars: 48hrs after GATA-1 transgene transfection. Data are relative to control antibody IPs (Y axis). T-test significance: p
    H3k9ac S10ph, supplied by Abcam, used in various techniques. Bioz Stars score: 76/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    Abcam chip grade h3k9ac
    Anti-H3K64ac antibody validation. ( A ) Western blot analysis with H3K64ac antibody (left panel) on nuclear extracts from untreated (−) or Na-butyrate-treated (+) HeLa cells. Ponceau staining is shown as loading control (right panel). ( B ), ( C ), ( D ), and ( E ) Immuno-dot blots showing specific reactivity of the H3K64ac antibody. Aliquots of indicated pmoles of linear H3 peptides were spotted on the membrane. Of note, detection of peptides in ( E ) has been checked with <t>H3K9ac,</t> H3K18ac, and H3K27ac specific antibodies. ( F ) Peptide competition assay of H3K64ac immunoblot. H3K64ac antibody was pre-adsorbed with 50 pmoles/ml of indicated peptides. Ponceau staining is shown as loading control (bottom panel). ( G ) The H3K64ac antibody detects globular, tailless H3. Nucleosomes from NIH3T3 cells were incubated with no (−) or increasing amounts of trypsin to cleave the histone tails and immunoblotted as indicated for H3K9ac, H3K18ac, H3K27ac, H3K64ac and H3. The position of full-length and tailless H3 is indicated and loading was controlled by ponceau staining. ( H ) Peptide competition assay in immunofluorescence. MEFs were treated overnight with Na-butyrate (NaB) and then stained with H3K64ac antibody, pre-incubated with 100 pmoles/ml of either unmodified or acetylated K64 peptide. ( I ) Immunoblot analysis of H3K64 acetylation state in additional cell lines (Drosophila S2, mouse MEFs, human HeLa) with H3K64ac antibody. Inhibition of HDACs by Na-butyrate treatment increases acetylation levels. Ponceau staining is shown as loading control (bottom panel). DOI: http://dx.doi.org/10.7554/eLife.01632.005
    Chip Grade H3k9ac, supplied by Abcam, used in various techniques. Bioz Stars score: 76/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Abcam h3k9ac k14ac
    Anti-H3K64ac antibody validation. ( A ) Western blot analysis with H3K64ac antibody (left panel) on nuclear extracts from untreated (−) or Na-butyrate-treated (+) HeLa cells. Ponceau staining is shown as loading control (right panel). ( B ), ( C ), ( D ), and ( E ) Immuno-dot blots showing specific reactivity of the H3K64ac antibody. Aliquots of indicated pmoles of linear H3 peptides were spotted on the membrane. Of note, detection of peptides in ( E ) has been checked with <t>H3K9ac,</t> H3K18ac, and H3K27ac specific antibodies. ( F ) Peptide competition assay of H3K64ac immunoblot. H3K64ac antibody was pre-adsorbed with 50 pmoles/ml of indicated peptides. Ponceau staining is shown as loading control (bottom panel). ( G ) The H3K64ac antibody detects globular, tailless H3. Nucleosomes from NIH3T3 cells were incubated with no (−) or increasing amounts of trypsin to cleave the histone tails and immunoblotted as indicated for H3K9ac, H3K18ac, H3K27ac, H3K64ac and H3. The position of full-length and tailless H3 is indicated and loading was controlled by ponceau staining. ( H ) Peptide competition assay in immunofluorescence. MEFs were treated overnight with Na-butyrate (NaB) and then stained with H3K64ac antibody, pre-incubated with 100 pmoles/ml of either unmodified or acetylated K64 peptide. ( I ) Immunoblot analysis of H3K64 acetylation state in additional cell lines (Drosophila S2, mouse MEFs, human HeLa) with H3K64ac antibody. Inhibition of HDACs by Na-butyrate treatment increases acetylation levels. Ponceau staining is shown as loading control (bottom panel). DOI: http://dx.doi.org/10.7554/eLife.01632.005
    H3k9ac K14ac, supplied by Abcam, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam primary antibodies against h3k9ac
    (A ) Western blot of total protein samples of Ada2b d842 mutant, w 1118 control, and Ada2bEGFP transgene carrier Ada2b d842 animals. The labels are P: pupa, L: larvae Ada2b resc : Ada2bEGFP transgene carrier Ada2b d842 and as indicated. The Mw of the two dADA2b isoforms are indicated. Note that in Ada2b resc the EGFP tag attached to the C-terminus of the larger dADA2b isoform increases its size. At the bottom on the left panel the same filter developed with alpha-tubulin-specific Ab as loading control is shown. ( B ) Western blot of total protein samples of Ada2b d842 mutants, w 1118 control and Ada2bEGFP transgene carrier Ada2b d842 animals developed with histone <t>H3K9ac-specific</t> antibody. Labels, genotypes and developmental stages are as indicate, and as in (A). On the bottom: the same filters developed with anti-histone H3 antibody. ( C ) Immunostaining of salivary glands (top) and of polytene chromosomes (bottom) of late third instar Ada2b d842 , control ( w 1118 ) and Ada2bEGFP transgene carrier Ada2b d842 larvae with H3K9ac- and H3K14ac-specific Abs. Pol II-specific staining (Ab: 7G5) of the same polytene chromosomes is shown as staining controls. ( D ) Accumulation of H3K9ac is not detectable in wild type ( w 1118 ) heat-shock puff (top). Puff is formed in the absence of H3K9ac in Ada2b d842 mutant (bottom). Puffs formed at the 93D cytological region are indicated by arrow as an example. Red: H3K9ac-specific, green: Pol II-specific Ab (H14) staining.
    Primary Antibodies Against H3k9ac, supplied by Abcam, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam lysine 9
    The histone 3 <t>lysine</t> 9 monomethylation (H3K9me1) level was analyzed as the mean gray value of hMSCs cultured on the MARC chip for 7 days. All data shown as average ± SD. Comparison of experimental replicas (N = 4) with at least 100 cells were analyzed in each of the experimental replica on each pattern type. * p represents statistical significance with p ≤ 0.05, ** p ≤ 0.01. Refer to Table 1 for abbreviations.
    Lysine 9, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 161 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Active Motif chromatin immunoprecipitation antibodies
    The histone 3 <t>lysine</t> 9 monomethylation (H3K9me1) level was analyzed as the mean gray value of hMSCs cultured on the MARC chip for 7 days. All data shown as average ± SD. Comparison of experimental replicas (N = 4) with at least 100 cells were analyzed in each of the experimental replica on each pattern type. * p represents statistical significance with p ≤ 0.05, ** p ≤ 0.01. Refer to Table 1 for abbreviations.
    Chromatin Immunoprecipitation Antibodies, supplied by Active Motif, used in various techniques. Bioz Stars score: 79/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam h3k9ac specific antibody
    (A ) Western blot of total protein samples of Ada2b d842 mutant, w 1118 control, and Ada2bEGFP transgene carrier Ada2b d842 animals. The labels are P: pupa, L: larvae Ada2b resc : Ada2bEGFP transgene carrier Ada2b d842 and as indicated. The Mw of the two dADA2b isoforms are indicated. Note that in Ada2b resc the EGFP tag attached to the C-terminus of the larger dADA2b isoform increases its size. At the bottom on the left panel the same filter developed with alpha-tubulin-specific Ab as loading control is shown. ( B ) Western blot of total protein samples of Ada2b d842 mutants, w 1118 control and Ada2bEGFP transgene carrier Ada2b d842 animals developed with histone <t>H3K9ac-specific</t> antibody. Labels, genotypes and developmental stages are as indicate, and as in (A). On the bottom: the same filters developed with anti-histone H3 antibody. ( C ) Immunostaining of salivary glands (top) and of polytene chromosomes (bottom) of late third instar Ada2b d842 , control ( w 1118 ) and Ada2bEGFP transgene carrier Ada2b d842 larvae with H3K9ac- and H3K14ac-specific Abs. Pol II-specific staining (Ab: 7G5) of the same polytene chromosomes is shown as staining controls. ( D ) Accumulation of H3K9ac is not detectable in wild type ( w 1118 ) heat-shock puff (top). Puff is formed in the absence of H3K9ac in Ada2b d842 mutant (bottom). Puffs formed at the 93D cytological region are indicated by arrow as an example. Red: H3K9ac-specific, green: Pol II-specific Ab (H14) staining.
    H3k9ac Specific Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 76/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam histone modifications
    (A ) Western blot of total protein samples of Ada2b d842 mutant, w 1118 control, and Ada2bEGFP transgene carrier Ada2b d842 animals. The labels are P: pupa, L: larvae Ada2b resc : Ada2bEGFP transgene carrier Ada2b d842 and as indicated. The Mw of the two dADA2b isoforms are indicated. Note that in Ada2b resc the EGFP tag attached to the C-terminus of the larger dADA2b isoform increases its size. At the bottom on the left panel the same filter developed with alpha-tubulin-specific Ab as loading control is shown. ( B ) Western blot of total protein samples of Ada2b d842 mutants, w 1118 control and Ada2bEGFP transgene carrier Ada2b d842 animals developed with histone <t>H3K9ac-specific</t> antibody. Labels, genotypes and developmental stages are as indicate, and as in (A). On the bottom: the same filters developed with anti-histone H3 antibody. ( C ) Immunostaining of salivary glands (top) and of polytene chromosomes (bottom) of late third instar Ada2b d842 , control ( w 1118 ) and Ada2bEGFP transgene carrier Ada2b d842 larvae with H3K9ac- and H3K14ac-specific Abs. Pol II-specific staining (Ab: 7G5) of the same polytene chromosomes is shown as staining controls. ( D ) Accumulation of H3K9ac is not detectable in wild type ( w 1118 ) heat-shock puff (top). Puff is formed in the absence of H3K9ac in Ada2b d842 mutant (bottom). Puffs formed at the 93D cytological region are indicated by arrow as an example. Red: H3K9ac-specific, green: Pol II-specific Ab (H14) staining.
    Histone Modifications, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 219 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Abcam histone h3k9ac
    (A ) Western blot of total protein samples of Ada2b d842 mutant, w 1118 control, and Ada2bEGFP transgene carrier Ada2b d842 animals. The labels are P: pupa, L: larvae Ada2b resc : Ada2bEGFP transgene carrier Ada2b d842 and as indicated. The Mw of the two dADA2b isoforms are indicated. Note that in Ada2b resc the EGFP tag attached to the C-terminus of the larger dADA2b isoform increases its size. At the bottom on the left panel the same filter developed with alpha-tubulin-specific Ab as loading control is shown. ( B ) Western blot of total protein samples of Ada2b d842 mutants, w 1118 control and Ada2bEGFP transgene carrier Ada2b d842 animals developed with histone <t>H3K9ac-specific</t> antibody. Labels, genotypes and developmental stages are as indicate, and as in (A). On the bottom: the same filters developed with anti-histone H3 antibody. ( C ) Immunostaining of salivary glands (top) and of polytene chromosomes (bottom) of late third instar Ada2b d842 , control ( w 1118 ) and Ada2bEGFP transgene carrier Ada2b d842 larvae with H3K9ac- and H3K14ac-specific Abs. Pol II-specific staining (Ab: 7G5) of the same polytene chromosomes is shown as staining controls. ( D ) Accumulation of H3K9ac is not detectable in wild type ( w 1118 ) heat-shock puff (top). Puff is formed in the absence of H3K9ac in Ada2b d842 mutant (bottom). Puffs formed at the 93D cytological region are indicated by arrow as an example. Red: H3K9ac-specific, green: Pol II-specific Ab (H14) staining.
    Histone H3k9ac, supplied by Abcam, used in various techniques. Bioz Stars score: 83/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam histone h3k9ac antibody
    (A ) Western blot of total protein samples of Ada2b d842 mutant, w 1118 control, and Ada2bEGFP transgene carrier Ada2b d842 animals. The labels are P: pupa, L: larvae Ada2b resc : Ada2bEGFP transgene carrier Ada2b d842 and as indicated. The Mw of the two dADA2b isoforms are indicated. Note that in Ada2b resc the EGFP tag attached to the C-terminus of the larger dADA2b isoform increases its size. At the bottom on the left panel the same filter developed with alpha-tubulin-specific Ab as loading control is shown. ( B ) Western blot of total protein samples of Ada2b d842 mutants, w 1118 control and Ada2bEGFP transgene carrier Ada2b d842 animals developed with histone <t>H3K9ac-specific</t> antibody. Labels, genotypes and developmental stages are as indicate, and as in (A). On the bottom: the same filters developed with anti-histone H3 antibody. ( C ) Immunostaining of salivary glands (top) and of polytene chromosomes (bottom) of late third instar Ada2b d842 , control ( w 1118 ) and Ada2bEGFP transgene carrier Ada2b d842 larvae with H3K9ac- and H3K14ac-specific Abs. Pol II-specific staining (Ab: 7G5) of the same polytene chromosomes is shown as staining controls. ( D ) Accumulation of H3K9ac is not detectable in wild type ( w 1118 ) heat-shock puff (top). Puff is formed in the absence of H3K9ac in Ada2b d842 mutant (bottom). Puffs formed at the 93D cytological region are indicated by arrow as an example. Red: H3K9ac-specific, green: Pol II-specific Ab (H14) staining.
    Histone H3k9ac Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 76/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Abcam euchromatin marker k9 acetylated histone h3
    (A ) Western blot of total protein samples of Ada2b d842 mutant, w 1118 control, and Ada2bEGFP transgene carrier Ada2b d842 animals. The labels are P: pupa, L: larvae Ada2b resc : Ada2bEGFP transgene carrier Ada2b d842 and as indicated. The Mw of the two dADA2b isoforms are indicated. Note that in Ada2b resc the EGFP tag attached to the C-terminus of the larger dADA2b isoform increases its size. At the bottom on the left panel the same filter developed with alpha-tubulin-specific Ab as loading control is shown. ( B ) Western blot of total protein samples of Ada2b d842 mutants, w 1118 control and Ada2bEGFP transgene carrier Ada2b d842 animals developed with histone <t>H3K9ac-specific</t> antibody. Labels, genotypes and developmental stages are as indicate, and as in (A). On the bottom: the same filters developed with anti-histone H3 antibody. ( C ) Immunostaining of salivary glands (top) and of polytene chromosomes (bottom) of late third instar Ada2b d842 , control ( w 1118 ) and Ada2bEGFP transgene carrier Ada2b d842 larvae with H3K9ac- and H3K14ac-specific Abs. Pol II-specific staining (Ab: 7G5) of the same polytene chromosomes is shown as staining controls. ( D ) Accumulation of H3K9ac is not detectable in wild type ( w 1118 ) heat-shock puff (top). Puff is formed in the absence of H3K9ac in Ada2b d842 mutant (bottom). Puffs formed at the 93D cytological region are indicated by arrow as an example. Red: H3K9ac-specific, green: Pol II-specific Ab (H14) staining.
    Euchromatin Marker K9 Acetylated Histone H3, supplied by Abcam, used in various techniques. Bioz Stars score: 79/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abcam rabbit anti h3k9ac
    Identification of positively stained and negative tumour cells using the Ariol system. The Ariol system trainer overlay shows correct identification of positive (yellow dots) and negative (blue dots) cells on tumour cores. TMA slides were scanned using a × 20 magnification. Shown for both <t>H3K9Ac</t> and H3K27me3 are highly positive tumour cores ( A and D ), tumour cores with both positive and negative cells ( B and E ) and negative tumour cores ( C and F ). The Ariol system was trained to identify positive and negative cells for each individual marker.
    Rabbit Anti H3k9ac, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam histone h3 lysine 9
    Identification of positively stained and negative tumour cells using the Ariol system. The Ariol system trainer overlay shows correct identification of positive (yellow dots) and negative (blue dots) cells on tumour cores. TMA slides were scanned using a × 20 magnification. Shown for both <t>H3K9Ac</t> and H3K27me3 are highly positive tumour cores ( A and D ), tumour cores with both positive and negative cells ( B and E ) and negative tumour cores ( C and F ). The Ariol system was trained to identify positive and negative cells for each individual marker.
    Histone H3 Lysine 9, supplied by Abcam, used in various techniques. Bioz Stars score: 84/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit polyclonal anti h3k9ac antibodies
    Identification of positively stained and negative tumour cells using the Ariol system. The Ariol system trainer overlay shows correct identification of positive (yellow dots) and negative (blue dots) cells on tumour cores. TMA slides were scanned using a × 20 magnification. Shown for both <t>H3K9Ac</t> and H3K27me3 are highly positive tumour cores ( A and D ), tumour cores with both positive and negative cells ( B and E ) and negative tumour cores ( C and F ). The Ariol system was trained to identify positive and negative cells for each individual marker.
    Rabbit Polyclonal Anti H3k9ac Antibodies, supplied by Abcam, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti h3k9 acetylation
    Identification of positively stained and negative tumour cells using the Ariol system. The Ariol system trainer overlay shows correct identification of positive (yellow dots) and negative (blue dots) cells on tumour cores. TMA slides were scanned using a × 20 magnification. Shown for both <t>H3K9Ac</t> and H3K27me3 are highly positive tumour cores ( A and D ), tumour cores with both positive and negative cells ( B and E ) and negative tumour cores ( C and F ). The Ariol system was trained to identify positive and negative cells for each individual marker.
    Rabbit Anti H3k9 Acetylation, supplied by Abcam, used in various techniques. Bioz Stars score: 80/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti h3k9ac monoclonal antibody
    Identification of positively stained and negative tumour cells using the Ariol system. The Ariol system trainer overlay shows correct identification of positive (yellow dots) and negative (blue dots) cells on tumour cores. TMA slides were scanned using a × 20 magnification. Shown for both <t>H3K9Ac</t> and H3K27me3 are highly positive tumour cores ( A and D ), tumour cores with both positive and negative cells ( B and E ) and negative tumour cores ( C and F ). The Ariol system was trained to identify positive and negative cells for each individual marker.
    Anti H3k9ac Monoclonal Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 84/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abcam histone modification antibodies
    Identification of positively stained and negative tumour cells using the Ariol system. The Ariol system trainer overlay shows correct identification of positive (yellow dots) and negative (blue dots) cells on tumour cores. TMA slides were scanned using a × 20 magnification. Shown for both <t>H3K9Ac</t> and H3K27me3 are highly positive tumour cores ( A and D ), tumour cores with both positive and negative cells ( B and E ) and negative tumour cores ( C and F ). The Ariol system was trained to identify positive and negative cells for each individual marker.
    Histone Modification Antibodies, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Abcam rabbit h3k9ac
    With the intrathecal delivery method used, HDACI did not appear to have a measurable effect on dorsal root ganglion (DRG) acetylation, suggesting that the mechanism of action was mostly central. (A) Representative Western blot of single rat L5 DRG after nerve transection and intrathecal vehicle or HDACi treatment. Total H3 was used as a loading control for acetylated H3K9. (B) Quantification of global <t>H3K9ac</t> revealed no difference between vehicle and HDACI treatment groups ( n = 4, P = not significant [n.s.]).
    Rabbit H3k9ac, supplied by Abcam, used in various techniques. Bioz Stars score: 84/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abcam histone h3
    Histone modification at the RARβ2-promoter region induced by HDAC inhibitor and ATRA treatment. (A) SAHA (10 µmol/L) treatment for 48 h, either alone or in combination with ATRA (1 µmol/L), strongly induced the hyperacetylation of <t>histone</t> H3 and restored RARβ2 expression in HeLa, SiHa, CaSki, and C33A cells. (B) Schematic of the RARβ2-promoter region. The exons of RARβ2 are represented by black boxes. The arrow indicates the transcription-initiation site. The PCR region (−168 to +22) for the ChIP assay included the core region of the RARE, the TATA box, and 22 bp of exon 1. (C) Representative ChIP-PCR data. PCR products were visualized via a 1% agarose gel stained with ethidium bromide. (D) DNA samples from the anti-H3K9ac IP as well as the input material and the mock immunoprecipitation samples were quantified by real-time PCR. Treatment with either 10 µmol/L SAHA or 3 mmol/L VPA led to a significant increase in RARβ2-RARE enrichment. The greatest increase in RARβ2-RARE enrichment was observed with the combination treatment. * P
    Histone H3, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 5233 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit polyclonal antibodies
    Histone modification at the RARβ2-promoter region induced by HDAC inhibitor and ATRA treatment. (A) SAHA (10 µmol/L) treatment for 48 h, either alone or in combination with ATRA (1 µmol/L), strongly induced the hyperacetylation of <t>histone</t> H3 and restored RARβ2 expression in HeLa, SiHa, CaSki, and C33A cells. (B) Schematic of the RARβ2-promoter region. The exons of RARβ2 are represented by black boxes. The arrow indicates the transcription-initiation site. The PCR region (−168 to +22) for the ChIP assay included the core region of the RARE, the TATA box, and 22 bp of exon 1. (C) Representative ChIP-PCR data. PCR products were visualized via a 1% agarose gel stained with ethidium bromide. (D) DNA samples from the anti-H3K9ac IP as well as the input material and the mock immunoprecipitation samples were quantified by real-time PCR. Treatment with either 10 µmol/L SAHA or 3 mmol/L VPA led to a significant increase in RARβ2-RARE enrichment. The greatest increase in RARβ2-RARE enrichment was observed with the combination treatment. * P
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    Abcam rabbit monoclonal anti h3k9ac
    Histone modification at the RARβ2-promoter region induced by HDAC inhibitor and ATRA treatment. (A) SAHA (10 µmol/L) treatment for 48 h, either alone or in combination with ATRA (1 µmol/L), strongly induced the hyperacetylation of <t>histone</t> H3 and restored RARβ2 expression in HeLa, SiHa, CaSki, and C33A cells. (B) Schematic of the RARβ2-promoter region. The exons of RARβ2 are represented by black boxes. The arrow indicates the transcription-initiation site. The PCR region (−168 to +22) for the ChIP assay included the core region of the RARE, the TATA box, and 22 bp of exon 1. (C) Representative ChIP-PCR data. PCR products were visualized via a 1% agarose gel stained with ethidium bromide. (D) DNA samples from the anti-H3K9ac IP as well as the input material and the mock immunoprecipitation samples were quantified by real-time PCR. Treatment with either 10 µmol/L SAHA or 3 mmol/L VPA led to a significant increase in RARβ2-RARE enrichment. The greatest increase in RARβ2-RARE enrichment was observed with the combination treatment. * P
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    Image Search Results


    BATF3 Inhibits Histone Acetylation at the Foxp3 Locus under iTreg-Polarizing Conditions (A) Immunoblot analysis of phosphorylated Smad3 and Stat5 in the nucleus of naive WT or Batf3-Tg CD4 + T cells at indicated hours upon iTreg polarization. (B) Quantitation as well as statistical analysis of the Smad3 and Stat5 expression in western blot experiments. (C and D) FACS sorted naive CD4 + Foxp3 − T cells (C and D) from WT or Batf3 -Tg Foxp3 -EGFP reporter mice were cultured under the iTreg-polarizing condition (with TGF-β and IL-2) or nonpolarizing condition conditions (without TGF-β and IL-2) for 48 hr. Shown are ChIP analysis of H3Ac, H4Ac (C), H3K9Ac, and H3K27Ac (D) at Foxp3 locus.

    Journal: Cell reports

    Article Title: OX40 Costimulation Inhibits Foxp3 Expression and Treg Induction via BATF3-Dependent and Independent Mechanisms

    doi: 10.1016/j.celrep.2018.06.052

    Figure Lengend Snippet: BATF3 Inhibits Histone Acetylation at the Foxp3 Locus under iTreg-Polarizing Conditions (A) Immunoblot analysis of phosphorylated Smad3 and Stat5 in the nucleus of naive WT or Batf3-Tg CD4 + T cells at indicated hours upon iTreg polarization. (B) Quantitation as well as statistical analysis of the Smad3 and Stat5 expression in western blot experiments. (C and D) FACS sorted naive CD4 + Foxp3 − T cells (C and D) from WT or Batf3 -Tg Foxp3 -EGFP reporter mice were cultured under the iTreg-polarizing condition (with TGF-β and IL-2) or nonpolarizing condition conditions (without TGF-β and IL-2) for 48 hr. Shown are ChIP analysis of H3Ac, H4Ac (C), H3K9Ac, and H3K27Ac (D) at Foxp3 locus.

    Article Snippet: Anti-H3Ac (39139), anti-H4Ac (39243), anti-H3K27Ac (39133; all from Active Motif), anti-H3K9Ac (ab4441; Abcam), and anti-Flag (F1804; Sigma-Aldrich) were used for the immunoprecipitation of chromatin with an EZ ChIP kit according to the manufacturer’s instructions (Millipore).

    Techniques: Quantitation Assay, Expressing, Western Blot, FACS, Mouse Assay, Cell Culture, Chromatin Immunoprecipitation

    MEG3 inhibits C-myc and CyclinD1 dependent on PKM2. A Co-immunoprecipitation (IP) with anti-PKM2 followed by Western blotting with Histone H3 in the Hep3B cell lines transfected with pCMV6-A-GFP, pCMV6-A-GFP-MEG3, respectively. IgG IP as negative control. INPUT refers to Western blotting with Histone H3. B Western blotting analysis with anti-pH3T11, anti- H3K9me3, anti-H3K9Ac in liver cancer cells Hep3B cell lines transfected with pCMV6-A-GFP, pCMV6-A-GFP-MEG3, pCMV6-A-GFP-MEG3 plus pcDNA3-PKM2, respectively. β-actin as an internal control. C Chromatin immunoprecipitation (CHIP) with anti-H3K9Ac followed by PCR with C-Myc promoter and CyclinD1 promoter primers in liver cancer cells Hep3B cell lines transfected with pCMV6-A-GFP, pCMV6-A-GFP-MEG3, respectively. IgG CHIP as negative control. C-Myc promoter and CyclinD1 promoter as INPUT. D Western blotting analysis with anti-C-myc, anti- cyclinD1 in liver cancer cells Hep3B cell lines transfected with pCMV6-A-GFP, pCMV6-A-GFP-MEG3, pCMV6-A-GFP-MEG3 plus pcDNA3-PKM2, respectively. β-actin as an internal control

    Journal: Cell Death & Disease

    Article Title: Long noncoding RNA MEG3 suppresses liver cancer cells growth through inhibiting β-catenin by activating PKM2 and inactivating PTEN

    doi: 10.1038/s41419-018-0305-7

    Figure Lengend Snippet: MEG3 inhibits C-myc and CyclinD1 dependent on PKM2. A Co-immunoprecipitation (IP) with anti-PKM2 followed by Western blotting with Histone H3 in the Hep3B cell lines transfected with pCMV6-A-GFP, pCMV6-A-GFP-MEG3, respectively. IgG IP as negative control. INPUT refers to Western blotting with Histone H3. B Western blotting analysis with anti-pH3T11, anti- H3K9me3, anti-H3K9Ac in liver cancer cells Hep3B cell lines transfected with pCMV6-A-GFP, pCMV6-A-GFP-MEG3, pCMV6-A-GFP-MEG3 plus pcDNA3-PKM2, respectively. β-actin as an internal control. C Chromatin immunoprecipitation (CHIP) with anti-H3K9Ac followed by PCR with C-Myc promoter and CyclinD1 promoter primers in liver cancer cells Hep3B cell lines transfected with pCMV6-A-GFP, pCMV6-A-GFP-MEG3, respectively. IgG CHIP as negative control. C-Myc promoter and CyclinD1 promoter as INPUT. D Western blotting analysis with anti-C-myc, anti- cyclinD1 in liver cancer cells Hep3B cell lines transfected with pCMV6-A-GFP, pCMV6-A-GFP-MEG3, pCMV6-A-GFP-MEG3 plus pcDNA3-PKM2, respectively. β-actin as an internal control

    Article Snippet: Standard Western blotting procedures were used with the following antibodies: anti-PCNA (Santa Cruz, Biotech), anti-CTCF (Santa Cruz, Biotech), anti-CREB (Santa Cruz, Biotech), anti-RNA polII(Abcam), anti-H3K9me3 (Abcam), anti-H3K36me3 (Abcam), anti-PKM2 (Abcam), anti-ERK1/2 (Abcam), anti-pPKM2(ser37) (Cell signaling), anti-Histone H3 (Abcam), anti-H3K9Ac (Abcam), anti-METTL3 (Santa Cruz, Biotech), anti-AKT (Abcam), anti-pAKT (Abcam), anti-GSK3β(ser9), anti-GSK3β(Tyr216), anti-GSK3β (Santa Cruz, Biotech), anti-β-catenin (Santa Cruz, Biotech), anti-HA, anti-Skp2 (Santa Cruz, Biotech), anti-TCF4 (Santa Cruz, Biotech), anti-LEF (Santa Cruz, Biotech), anti-PTEN (Santa Cruz, Biotech), anti-LEF (Santa Cruz, Biotech), anti-TCF4 (Santa Cruz, Biotech), anti-C-myc (Santa Cruz, Biotech), and anti-cyclinD1 (Santa Cruz, Biotech).

    Techniques: Immunoprecipitation, Western Blot, Transfection, Negative Control, Chromatin Immunoprecipitation, Polymerase Chain Reaction

    YEATS2 is required for ATAC-dependent maintenance of histone H3K9ac on the ribosomal protein genes. a Western blot analysis of YEATS2 and H3 and H4 acetylation in control (shNT) and YEATS2 KD (shY2) cells. H3 and H4 were used as a loading control. The arrow indicates the band of YEATS2 protein. b Average genome-wide H3K9ac occupancy on the promoter (5 kb±TSS) of the ZZZ3-bound genes or non-ZZZ3-bound genes (others) in control (shNT) and YEATS2 KD (shY2) H1299 cells. c Genome-browser view of the H3K9ac and ZZZ3 ChIP-seq peaks on the indicated ribosomal protein genes in cells as in ( b ). d qPCR analysis of H3K9ac ChIP of the indicated ribosomal protein genes in cells as in ( b ). e qPCR analysis of ZZZ3 ChIP of the indicated ribosomal protein genes in cells as in ( b ). f Average ZZZ3 occupancy on the promoter (5 kb±TSS) of the ZZZ3-bound genes or non-ZZZ3-bound genes (others) in control (shNT) and YEATS2 KD (shY2) H1299 cells. In d and e , error bars indicate S.E.M. of at least three biological replicates. * p

    Journal: Nature Communications

    Article Title: YEATS2 links histone acetylation to tumorigenesis of non-small cell lung cancer

    doi: 10.1038/s41467-017-01173-4

    Figure Lengend Snippet: YEATS2 is required for ATAC-dependent maintenance of histone H3K9ac on the ribosomal protein genes. a Western blot analysis of YEATS2 and H3 and H4 acetylation in control (shNT) and YEATS2 KD (shY2) cells. H3 and H4 were used as a loading control. The arrow indicates the band of YEATS2 protein. b Average genome-wide H3K9ac occupancy on the promoter (5 kb±TSS) of the ZZZ3-bound genes or non-ZZZ3-bound genes (others) in control (shNT) and YEATS2 KD (shY2) H1299 cells. c Genome-browser view of the H3K9ac and ZZZ3 ChIP-seq peaks on the indicated ribosomal protein genes in cells as in ( b ). d qPCR analysis of H3K9ac ChIP of the indicated ribosomal protein genes in cells as in ( b ). e qPCR analysis of ZZZ3 ChIP of the indicated ribosomal protein genes in cells as in ( b ). f Average ZZZ3 occupancy on the promoter (5 kb±TSS) of the ZZZ3-bound genes or non-ZZZ3-bound genes (others) in control (shNT) and YEATS2 KD (shY2) H1299 cells. In d and e , error bars indicate S.E.M. of at least three biological replicates. * p

    Article Snippet: Keck Facility at Yale University or CPC Scientific Inc. Anti-histone antibodies including anti-H3 (Ab1791, WB 1:20000), anti-H3K9ac (Ab32129, WB 1:1000), anti-H3K14ac (Ab52946, WB 1:1000), anti-H3K27ac (Ab4729, WB 1:1000), anti-H4 (Ab731, WB 1:5000), and anti-HDAC1(ab19845, WB 1:1000) antibodies were obtained from Abcam; anti-H3K9ac (61251) and anti-H4K16ac (39167 WB 1:1000) from Active Motif; anti-H4 tetra-acetyl antibody (06-598, WB 1:5000) from Millipore; anti-GCN5 (sc-20698, WB 1:2000), anti-PCAF (sc-13124, WB 1:200), anti-ADA3 (sc-98821, WB 1:1000), anti-ATAC2 (sc-398475, WB 1:1000), and anti-GST (sc-459, WB 1:1000) antibodies from Santa Cruz; anti-actin (A1978, WB 1:5000), anti-ZZZ3 (SAB4501106, WB 1:1000), and anti-tubulin (T8328, WB 1:5000) antibodies from Sigma; and anti-YEATS2 (24717-1-AP, WB 1:1000) antibody from ProteinTech.

    Techniques: Western Blot, Genome Wide, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    The YEATS domain of YEATS2 is required for ATAC-dependent ribosomal protein gene expression and tumor cell survival. a qPCR analysis of H3K9ac ChIP in the promoters of the indicated ribosomal protein genes in control (shNT) and YEATS2 KD (shY2) H1299 cells ectopically expressing shRNA-resistant WT YEATS2 or the indicated mutants. b qRT-PCR analysis of the expression of ribosomal protein genes in cells as in ( a ). In a and b , error bars indicate S.E.M. of at least three biological replicates. N.S. not significant; * p

    Journal: Nature Communications

    Article Title: YEATS2 links histone acetylation to tumorigenesis of non-small cell lung cancer

    doi: 10.1038/s41467-017-01173-4

    Figure Lengend Snippet: The YEATS domain of YEATS2 is required for ATAC-dependent ribosomal protein gene expression and tumor cell survival. a qPCR analysis of H3K9ac ChIP in the promoters of the indicated ribosomal protein genes in control (shNT) and YEATS2 KD (shY2) H1299 cells ectopically expressing shRNA-resistant WT YEATS2 or the indicated mutants. b qRT-PCR analysis of the expression of ribosomal protein genes in cells as in ( a ). In a and b , error bars indicate S.E.M. of at least three biological replicates. N.S. not significant; * p

    Article Snippet: Keck Facility at Yale University or CPC Scientific Inc. Anti-histone antibodies including anti-H3 (Ab1791, WB 1:20000), anti-H3K9ac (Ab32129, WB 1:1000), anti-H3K14ac (Ab52946, WB 1:1000), anti-H3K27ac (Ab4729, WB 1:1000), anti-H4 (Ab731, WB 1:5000), and anti-HDAC1(ab19845, WB 1:1000) antibodies were obtained from Abcam; anti-H3K9ac (61251) and anti-H4K16ac (39167 WB 1:1000) from Active Motif; anti-H4 tetra-acetyl antibody (06-598, WB 1:5000) from Millipore; anti-GCN5 (sc-20698, WB 1:2000), anti-PCAF (sc-13124, WB 1:200), anti-ADA3 (sc-98821, WB 1:1000), anti-ATAC2 (sc-398475, WB 1:1000), and anti-GST (sc-459, WB 1:1000) antibodies from Santa Cruz; anti-actin (A1978, WB 1:5000), anti-ZZZ3 (SAB4501106, WB 1:1000), and anti-tubulin (T8328, WB 1:5000) antibodies from Sigma; and anti-YEATS2 (24717-1-AP, WB 1:1000) antibody from ProteinTech.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, shRNA, Quantitative RT-PCR

    YEATS2 is amplified in NSCLC and is required for cancer cell growth and survival. a YEATS2 gene is frequently amplified in various types of human cancers. Data was obtained from the cBioPortal for Cancer Genomics. b Western blot analysis of YEATS2, GCN5, PCAF, HDAC1, and the indicated histone acetylation in NSCLC cell lines and immortalized “normal” lung fibroblast cell lines. Total H3 and actin are shown as loading control. The arrow indicates the band of YEATS2 protein. Relative H3K9ac, H3K14ac, and H3K27ac levels were quantified ( n = 3, mean ± s.e.m.). N.S. not significant; * p

    Journal: Nature Communications

    Article Title: YEATS2 links histone acetylation to tumorigenesis of non-small cell lung cancer

    doi: 10.1038/s41467-017-01173-4

    Figure Lengend Snippet: YEATS2 is amplified in NSCLC and is required for cancer cell growth and survival. a YEATS2 gene is frequently amplified in various types of human cancers. Data was obtained from the cBioPortal for Cancer Genomics. b Western blot analysis of YEATS2, GCN5, PCAF, HDAC1, and the indicated histone acetylation in NSCLC cell lines and immortalized “normal” lung fibroblast cell lines. Total H3 and actin are shown as loading control. The arrow indicates the band of YEATS2 protein. Relative H3K9ac, H3K14ac, and H3K27ac levels were quantified ( n = 3, mean ± s.e.m.). N.S. not significant; * p

    Article Snippet: Keck Facility at Yale University or CPC Scientific Inc. Anti-histone antibodies including anti-H3 (Ab1791, WB 1:20000), anti-H3K9ac (Ab32129, WB 1:1000), anti-H3K14ac (Ab52946, WB 1:1000), anti-H3K27ac (Ab4729, WB 1:1000), anti-H4 (Ab731, WB 1:5000), and anti-HDAC1(ab19845, WB 1:1000) antibodies were obtained from Abcam; anti-H3K9ac (61251) and anti-H4K16ac (39167 WB 1:1000) from Active Motif; anti-H4 tetra-acetyl antibody (06-598, WB 1:5000) from Millipore; anti-GCN5 (sc-20698, WB 1:2000), anti-PCAF (sc-13124, WB 1:200), anti-ADA3 (sc-98821, WB 1:1000), anti-ATAC2 (sc-398475, WB 1:1000), and anti-GST (sc-459, WB 1:1000) antibodies from Santa Cruz; anti-actin (A1978, WB 1:5000), anti-ZZZ3 (SAB4501106, WB 1:1000), and anti-tubulin (T8328, WB 1:5000) antibodies from Sigma; and anti-YEATS2 (24717-1-AP, WB 1:1000) antibody from ProteinTech.

    Techniques: Amplification, Western Blot

    NPY expression is epigenetically regulated and modulates β cell maturation. ( A – C ) ChIP analysis showing the enrichment of histone modifications ( A ) H3K9me3 (histone H3-lysine 9 trimethylation; repressive) and H3K9Ac (histone H3 lysine 9 acetylation; activating), ( B ) histone acetyl transferase CBP (CREB-binding protein) and histone deacteylase HDAC2, ( C ) along with control rabbit and mouse IgGs, to the Npy promoter region in β cells sorted from MIP-GFP mice at P5 and P30. These data show the epigenetic repression of the Npy promoter as a function of maturation. ( D ) Npy mRNA expression levels, normalized to the housekeeping gene CyclophilinA in β cells sorted from MIP-GFP mice, in which GFP expression is driven by insulin promoter at P5 and P30, showing reduced expression as a function of maturation. ( E ) A representative pancreatic section from wild-type, neonatal (P5) mice showing immunostaining for NPY (green), insulin (Ins; red), and Ki67 (cyan). Arrows mark replicating NPY + β cells. ( F ) Quantification of replication marker Ki67 in the NPY + and NPY – subpopulations of β cells in wild-type neonatal mice at P5 and P14. ( G ) Immunohistochemistry and ( H ) quantification for replication of β cells in islets isolated from neonatal (P5) mice treated with an siRNA targeting Npy or a control, scrambled siRNA. Immunostaining for replication marker Ki67 (red) and β cell marker Pdx1 (green) was used to measure β cell replication. Arrows mark Ki67 Pdx1 double-positive cells. ( I – K ) Npy mRNA levels and static incubation glucose-stimulated insulin secretion (GSIS) assay in islets from ( I ) wild-type, ( J ) P5 pups, or ( K ) adult (2.5-month-old) mice, treated either with an siRNA targeting Npy or a scrambled (Scr) siRNA. Insulin secretion was measured at 2.8 mM glucose (basal) and 16.7 mM (stimulated) glucose and reported as a percentage of insulin content. Scale bar: 50 μm. For A – D , average of n = 3 independent sorts; each sort had 6–8 pups at P5 and 4 mice at P30. For E and F , n = 4 animals. For G – K , n = 3 independent experiments, with each replicate representing a pool of islets from 6–8 pups. The error bars represent SEM of the mean. * P

    Journal: JCI Insight

    Article Title: Neuropeptide Y expression marks partially differentiated β cells in mice and humans

    doi: 10.1172/jci.insight.94005

    Figure Lengend Snippet: NPY expression is epigenetically regulated and modulates β cell maturation. ( A – C ) ChIP analysis showing the enrichment of histone modifications ( A ) H3K9me3 (histone H3-lysine 9 trimethylation; repressive) and H3K9Ac (histone H3 lysine 9 acetylation; activating), ( B ) histone acetyl transferase CBP (CREB-binding protein) and histone deacteylase HDAC2, ( C ) along with control rabbit and mouse IgGs, to the Npy promoter region in β cells sorted from MIP-GFP mice at P5 and P30. These data show the epigenetic repression of the Npy promoter as a function of maturation. ( D ) Npy mRNA expression levels, normalized to the housekeeping gene CyclophilinA in β cells sorted from MIP-GFP mice, in which GFP expression is driven by insulin promoter at P5 and P30, showing reduced expression as a function of maturation. ( E ) A representative pancreatic section from wild-type, neonatal (P5) mice showing immunostaining for NPY (green), insulin (Ins; red), and Ki67 (cyan). Arrows mark replicating NPY + β cells. ( F ) Quantification of replication marker Ki67 in the NPY + and NPY – subpopulations of β cells in wild-type neonatal mice at P5 and P14. ( G ) Immunohistochemistry and ( H ) quantification for replication of β cells in islets isolated from neonatal (P5) mice treated with an siRNA targeting Npy or a control, scrambled siRNA. Immunostaining for replication marker Ki67 (red) and β cell marker Pdx1 (green) was used to measure β cell replication. Arrows mark Ki67 Pdx1 double-positive cells. ( I – K ) Npy mRNA levels and static incubation glucose-stimulated insulin secretion (GSIS) assay in islets from ( I ) wild-type, ( J ) P5 pups, or ( K ) adult (2.5-month-old) mice, treated either with an siRNA targeting Npy or a scrambled (Scr) siRNA. Insulin secretion was measured at 2.8 mM glucose (basal) and 16.7 mM (stimulated) glucose and reported as a percentage of insulin content. Scale bar: 50 μm. For A – D , average of n = 3 independent sorts; each sort had 6–8 pups at P5 and 4 mice at P30. For E and F , n = 4 animals. For G – K , n = 3 independent experiments, with each replicate representing a pool of islets from 6–8 pups. The error bars represent SEM of the mean. * P

    Article Snippet: The following antibodies were used: H3K9me3 (EMD Millipore 07-442), H3K9Ac (Abcam AB12179), CBP (Diagenode C15410224-25), HDAC2 (Abcam AB12169), and control mouse and rabbit IgGs (Diagenode; KCH-819-015 and KCH-504-250, respectively).

    Techniques: Expressing, Chromatin Immunoprecipitation, Binding Assay, Mouse Assay, Immunostaining, Marker, Immunohistochemistry, Isolation, Incubation

    NPY is reexpressed in the β cells preceding disease onset in mouse models of diabetes. ( A – D ) Immunostaining and quantification of NPY expression in pancreatic sections from db/db (age: 6 weeks; A and B ) and NOD (age: 8 weeks; C and D ) mice, with age-matched controls. Representative pancreatic sections stained for NPY (red), somatostatin (SS; green), and insulin (Ins; cyan) are shown, with DAPI marking nuclei in blue. ( E ) Transcript levels for Npy in islets from wild-type adult (2.5-month-old) mice treated with H 2 O 2 or vehicle control. ( F ) ChIP analysis showing the levels of histone modifications H3K9me3 (histone H3-lysine 9 trimethylation; repressive) and H3K9Ac (histone H3 lysine 9 acetylation; activating) at the Npy promoter region in islets from db/db (age = 6 weeks; left) and NOD (age: 8 weeks; right) mice compared with age-matched controls, showing that the Npy promoter is active in these diabetic models. ( G ) Static incubation glucose-stimulated insulin secretion (GSIS) assay in islets from 6-week-old db/db mice, treated either with an siRNA targeting Npy or a control scrambled (Scr) siRNA. Insulin secretion was measured at 2.8 mM glucose (basal) and 16.7 mM (stimulated) glucose and is reported as a percentage of insulin content. These data show an improvement of GSIS in islets from prediabetic db/db mice upon treatment with Npy siRNA. Scale bar: 50 μm. For A – D , n = 4 animals per group. For E – G , n = 3 independent experiments, with each replicate representing a pool of islets from 3 mice. The error bars represent SEM of the mean. * P

    Journal: JCI Insight

    Article Title: Neuropeptide Y expression marks partially differentiated β cells in mice and humans

    doi: 10.1172/jci.insight.94005

    Figure Lengend Snippet: NPY is reexpressed in the β cells preceding disease onset in mouse models of diabetes. ( A – D ) Immunostaining and quantification of NPY expression in pancreatic sections from db/db (age: 6 weeks; A and B ) and NOD (age: 8 weeks; C and D ) mice, with age-matched controls. Representative pancreatic sections stained for NPY (red), somatostatin (SS; green), and insulin (Ins; cyan) are shown, with DAPI marking nuclei in blue. ( E ) Transcript levels for Npy in islets from wild-type adult (2.5-month-old) mice treated with H 2 O 2 or vehicle control. ( F ) ChIP analysis showing the levels of histone modifications H3K9me3 (histone H3-lysine 9 trimethylation; repressive) and H3K9Ac (histone H3 lysine 9 acetylation; activating) at the Npy promoter region in islets from db/db (age = 6 weeks; left) and NOD (age: 8 weeks; right) mice compared with age-matched controls, showing that the Npy promoter is active in these diabetic models. ( G ) Static incubation glucose-stimulated insulin secretion (GSIS) assay in islets from 6-week-old db/db mice, treated either with an siRNA targeting Npy or a control scrambled (Scr) siRNA. Insulin secretion was measured at 2.8 mM glucose (basal) and 16.7 mM (stimulated) glucose and is reported as a percentage of insulin content. These data show an improvement of GSIS in islets from prediabetic db/db mice upon treatment with Npy siRNA. Scale bar: 50 μm. For A – D , n = 4 animals per group. For E – G , n = 3 independent experiments, with each replicate representing a pool of islets from 3 mice. The error bars represent SEM of the mean. * P

    Article Snippet: The following antibodies were used: H3K9me3 (EMD Millipore 07-442), H3K9Ac (Abcam AB12179), CBP (Diagenode C15410224-25), HDAC2 (Abcam AB12169), and control mouse and rabbit IgGs (Diagenode; KCH-819-015 and KCH-504-250, respectively).

    Techniques: Immunostaining, Expressing, Mouse Assay, Staining, Chromatin Immunoprecipitation, Incubation

    Panel A: Location of E4BP4 (E, green) and XFD1 (X, red) sites in non-coding DNA within the human APP gene and surrounding 50 kb DNA. The green and red vertical lines above or below the horizontal line indicate sites in the forward and reverse strand of DNA, respectively. The short yellow vertical bars indicate exons of APP. The first three exons are very close to one another near the transcription start site. Stars indicate the E4BP4 sites marked by H3K9Ac in chromatin immunoprecipitation (ChIP) assays in a human cell-line SHSY5Y expressing APP gene. The bent arrow indicates transcription start site of APP gene. The SHSY5Y cells were analyzed by ChIP using anti-H3K9Ac antibodies as described in Methods. Y-axis represents the amount of material present in anti-H3K9Ac ( Panel B ) or non-specific IgG ( Panel C ) immunoprecipitates compared to input chromatin used for the assay. Note the different scales for the two panels. Results shown are the average of three independent ChIP experiments, each of which was assayed in duplicates. Error bars represent standard deviation between experiments. Amplification from IkBα served as a positive control. The fold enrichment, over control IgG, of H3K9Ac activity at the APP intron 2 site (positive control) and the intron 4 sites are 180-fold versus 120-fold, respectively, as indicated at the top of Panel 6B.

    Journal: BMC Genomics

    Article Title: Using BAC transgenesis in zebrafish to identify regulatory sequences of the amyloid precursor protein gene in humans

    doi: 10.1186/1471-2164-13-451

    Figure Lengend Snippet: Panel A: Location of E4BP4 (E, green) and XFD1 (X, red) sites in non-coding DNA within the human APP gene and surrounding 50 kb DNA. The green and red vertical lines above or below the horizontal line indicate sites in the forward and reverse strand of DNA, respectively. The short yellow vertical bars indicate exons of APP. The first three exons are very close to one another near the transcription start site. Stars indicate the E4BP4 sites marked by H3K9Ac in chromatin immunoprecipitation (ChIP) assays in a human cell-line SHSY5Y expressing APP gene. The bent arrow indicates transcription start site of APP gene. The SHSY5Y cells were analyzed by ChIP using anti-H3K9Ac antibodies as described in Methods. Y-axis represents the amount of material present in anti-H3K9Ac ( Panel B ) or non-specific IgG ( Panel C ) immunoprecipitates compared to input chromatin used for the assay. Note the different scales for the two panels. Results shown are the average of three independent ChIP experiments, each of which was assayed in duplicates. Error bars represent standard deviation between experiments. Amplification from IkBα served as a positive control. The fold enrichment, over control IgG, of H3K9Ac activity at the APP intron 2 site (positive control) and the intron 4 sites are 180-fold versus 120-fold, respectively, as indicated at the top of Panel 6B.

    Article Snippet: The anti-H3K9Ac antibody was purchased from Abcam, Cambridge, MA.

    Techniques: Chromatin Immunoprecipitation, Expressing, Standard Deviation, Amplification, Positive Control, Activity Assay

    H3K9 methylation at the E-cadherin promoter is associated with TGF-β–induced EMT in HNSCC (A) HN4 cells were treated with TGF-β1 (5 ng/ml) for 3, 6, 9, 12 and 15 days, respectively; morphologic changes associated with EMT at 12 days are shown in the phase contrast images. Immunofluorescence (IF) staining for E-cadherin, N-cadherin and vimentin is presented with nuclei stained with DAPI (blue). Scale bar = 200 μm. (B) Migration of HN4 cells exposed to TGF-β (5 ng/ml) for 6 days before assay with representative images shown. Scale bar = 200 μm. (C) Graph demonstrates the mean ± SD percent of migrated cells from 3 separate experiments. (D) Western blot analysis of E-cadherin, N-cadherin and Snail in HN4 cells treated with TGF-β (5 ng/ml) for the indicated time periods. (E) ChIP analysis of H3K9me2 and H3K9Ac at the E-cadherin promoter of HN4 cells treated with TGF-β (5 ng/ml).

    Journal: Oncotarget

    Article Title: G9a is essential for EMT-mediated metastasis and maintenance of cancer stem cell-like characters in head and neck squamous cell carcinoma

    doi:

    Figure Lengend Snippet: H3K9 methylation at the E-cadherin promoter is associated with TGF-β–induced EMT in HNSCC (A) HN4 cells were treated with TGF-β1 (5 ng/ml) for 3, 6, 9, 12 and 15 days, respectively; morphologic changes associated with EMT at 12 days are shown in the phase contrast images. Immunofluorescence (IF) staining for E-cadherin, N-cadherin and vimentin is presented with nuclei stained with DAPI (blue). Scale bar = 200 μm. (B) Migration of HN4 cells exposed to TGF-β (5 ng/ml) for 6 days before assay with representative images shown. Scale bar = 200 μm. (C) Graph demonstrates the mean ± SD percent of migrated cells from 3 separate experiments. (D) Western blot analysis of E-cadherin, N-cadherin and Snail in HN4 cells treated with TGF-β (5 ng/ml) for the indicated time periods. (E) ChIP analysis of H3K9me2 and H3K9Ac at the E-cadherin promoter of HN4 cells treated with TGF-β (5 ng/ml).

    Article Snippet: G9a, H3K9me2, and H3k9Ac antibodies were from Abcam (Cambridge, MA).

    Techniques: Methylation, Immunofluorescence, Staining, Migration, Western Blot, Chromatin Immunoprecipitation

    The levels of histone H3K4me1, H3K4me2, H4K20me1, H3K9ac, H3K27ac, and H3K56ac in the livers of control rats and rats treated with furan. A, Representative immunoblots images of histone modifications in the livers of control rats and rats treated with furan. B, Densitometric analysis of the Western blot results is shown as percent change in histone modification level in the livers of furan-treated rats relative to that in the control group, which was assigned a value of 100%. Values are mean ± SD, n = 5. Asterisk (*) denotes a significant ( P

    Journal: Toxicological Sciences

    Article Title: Persistence of Furan-Induced Epigenetic Aberrations in the Livers of F344 Rats

    doi: 10.1093/toxsci/kfu313

    Figure Lengend Snippet: The levels of histone H3K4me1, H3K4me2, H4K20me1, H3K9ac, H3K27ac, and H3K56ac in the livers of control rats and rats treated with furan. A, Representative immunoblots images of histone modifications in the livers of control rats and rats treated with furan. B, Densitometric analysis of the Western blot results is shown as percent change in histone modification level in the livers of furan-treated rats relative to that in the control group, which was assigned a value of 100%. Values are mean ± SD, n = 5. Asterisk (*) denotes a significant ( P

    Article Snippet: Formaldehyde cross-linking and chromatin immunoprecipitation (ChIP) assays with primary antibodies against H3K9ac (Abcam, Cambridge, Massachusetts) were performed by using a ChIP Assay kit (Millipore Corporation, Billerica, Massachusetts).

    Techniques: Western Blot, Modification

    Nucleosome Modification Patterns (A) Schematic of annotation scheme for nucleosomes based on their position relative to transcribed units. Intergenic nucleosomes were assigned to the following categories: promoter region (anything upstream of a coding region), nucleosome immediately upstream to the TSS (“distal”), and the nucleosome immediately downstream of the TSS (“proximal”). Transcribed regions were separated into 5′, middle, and 3′ CDSs. Finally, to capture features of chromatin not associated with PolII genes, we independently classified nucleosomes associated with ARS sequences, tRNA genes, and Null (any other intergenic region). (B) Hierarchical clustering of 2,288 nucleosomes. Left panel: each row corresponds to a single nucleosome, and each column to a particular modification. Red (green) denotes hyper-acetylation (hypo-acetylation) in the first nine columns and relative level of methylation in the last three columns. Rows are sorted according to the dendogram built during clustering. PolII shows the PolII occupancy of the gene associated with the nucleosome in question. Right panel: each row corresponds to a nucleosome (matching the left panel), and each column corresponds to an annotation of the nucleosome according to the scheme of (A). A blue cell denotes a positive annotation of the nucleosome with the appropriate column label. Numbers indicate examples of clusters, as follows: (1) nucleosomes enriched for H3K9Ac, H3K14Ac, and H3K4Me3 that are mostly upstream of transcribed regions; (2) strongly hypo-acetylated nucleosomes, mostly at upstream regions or 3′ of coding regions; (3) nucleosomes acetylated at H4K8 and K16, and H2B K16 that are almost exclusively at the middle and 3′-ends of coding regions; and (4) hyper-acetylated and methylated nucleosomes that are mostly found at the 5′-end of coding regions. (C) The Pearson correlations of the 12 modification levels between different probes show that there are two tightly correlated groups of acetylations at specific residues. The first group consists of H2A K7; H3K9, K14, and K18; and H4K5 and K12. The second group consists of H2B K16; and H4K8 and K16. Mono- and di-methylation of H3K4 are correlated with the second group, while tri-methylation of H3K4 is correlated with the first group. (D) The percent of variance captured by using different number of components. The x -axis denotes the number of components, and the y -axis denotes the percent of the variance in the data explained by each components (blue bars) as well as the cumulative percentage explained (red bars). (E) Representation of all nucleosomes in two-dimensional modification space. In the left panel, each point represents a nucleosome plotted according to the relative level of the first principal component ( x -axis) and second principal component ( y -axis) for the modification pattern. The right panel is a three-dimensional plot showing density of points along the plane.

    Journal: PLoS Biology

    Article Title: Single-Nucleosome Mapping of Histone Modifications in S. cerevisiaeA Global View of DNA-Packing Proteins Cracks the Histone Code

    doi: 10.1371/journal.pbio.0030328

    Figure Lengend Snippet: Nucleosome Modification Patterns (A) Schematic of annotation scheme for nucleosomes based on their position relative to transcribed units. Intergenic nucleosomes were assigned to the following categories: promoter region (anything upstream of a coding region), nucleosome immediately upstream to the TSS (“distal”), and the nucleosome immediately downstream of the TSS (“proximal”). Transcribed regions were separated into 5′, middle, and 3′ CDSs. Finally, to capture features of chromatin not associated with PolII genes, we independently classified nucleosomes associated with ARS sequences, tRNA genes, and Null (any other intergenic region). (B) Hierarchical clustering of 2,288 nucleosomes. Left panel: each row corresponds to a single nucleosome, and each column to a particular modification. Red (green) denotes hyper-acetylation (hypo-acetylation) in the first nine columns and relative level of methylation in the last three columns. Rows are sorted according to the dendogram built during clustering. PolII shows the PolII occupancy of the gene associated with the nucleosome in question. Right panel: each row corresponds to a nucleosome (matching the left panel), and each column corresponds to an annotation of the nucleosome according to the scheme of (A). A blue cell denotes a positive annotation of the nucleosome with the appropriate column label. Numbers indicate examples of clusters, as follows: (1) nucleosomes enriched for H3K9Ac, H3K14Ac, and H3K4Me3 that are mostly upstream of transcribed regions; (2) strongly hypo-acetylated nucleosomes, mostly at upstream regions or 3′ of coding regions; (3) nucleosomes acetylated at H4K8 and K16, and H2B K16 that are almost exclusively at the middle and 3′-ends of coding regions; and (4) hyper-acetylated and methylated nucleosomes that are mostly found at the 5′-end of coding regions. (C) The Pearson correlations of the 12 modification levels between different probes show that there are two tightly correlated groups of acetylations at specific residues. The first group consists of H2A K7; H3K9, K14, and K18; and H4K5 and K12. The second group consists of H2B K16; and H4K8 and K16. Mono- and di-methylation of H3K4 are correlated with the second group, while tri-methylation of H3K4 is correlated with the first group. (D) The percent of variance captured by using different number of components. The x -axis denotes the number of components, and the y -axis denotes the percent of the variance in the data explained by each components (blue bars) as well as the cumulative percentage explained (red bars). (E) Representation of all nucleosomes in two-dimensional modification space. In the left panel, each point represents a nucleosome plotted according to the relative level of the first principal component ( x -axis) and second principal component ( y -axis) for the modification pattern. The right panel is a three-dimensional plot showing density of points along the plane.

    Article Snippet: With the remainder, antibodies were added to each aliquot (20% of a 450-ml cell culture) in the following volumes: 25 μl anti-H3K4Me1 Ab (affinity purified; Abcam, Cambridge, Massachusetts, United States), 6 μl anti-H3K4Me2 Ab (affinity purified; Abcam), 6 μl anti-H3K4Me3 Ab (affinity purified; Abcam), 4 μl anti-H4K16Ac Ab (whole antiserum; Abcam), 9 μl anti-H4K5Ac Ab (whole antiserum; Abcam), 3 μl anti-H3K14Ac Ab (whole antiserum; Upstate Cell Signaling Solutions, Charlottesville, Virginia, United States), 3 μl anti-H2AK7Ac Ab (whole antiserum; Upstate), 2 μl, anti-H4K8Ac Ab (whole antiserum; Abcam), 15 μl, anti-H4K12Ac Ab (whole antiserum; Abcam), 25 μl anti-Ac Ab (whole antiserum; Abcam), 16 μl anti-H3K9Ac Ab (affinity purified; Abcam), 25 μl anti-H2BK16Ac (L) (whole antiserum; Abcam), and 3 μl anti-H3K18Ac Ab (whole antiserum; gift of M. Grunstein).

    Techniques: Modification, Methylation

    Repressive histone modifications following GATA-1 overexpression in AML-ELs. ChIP at the PU . 1 gene locus was carried out for the H3K9Me3, H3K27Me3, and H3K9Ac histone tail modifications in OCI-M2 (left) and K562 (right) cells. Grey bars: control cells, dark bars: 48hrs after GATA-1 transgene transfection. Data are relative to control antibody IPs (Y axis). T-test significance: p

    Journal: PLoS ONE

    Article Title: GATA-1 Inhibits PU.1 Gene via DNA and Histone H3K9 Methylation of Its Distal Enhancer in Erythroleukemia

    doi: 10.1371/journal.pone.0152234

    Figure Lengend Snippet: Repressive histone modifications following GATA-1 overexpression in AML-ELs. ChIP at the PU . 1 gene locus was carried out for the H3K9Me3, H3K27Me3, and H3K9Ac histone tail modifications in OCI-M2 (left) and K562 (right) cells. Grey bars: control cells, dark bars: 48hrs after GATA-1 transgene transfection. Data are relative to control antibody IPs (Y axis). T-test significance: p

    Article Snippet: IP-antibodies: GATA-1 (N6/sc265-Santa Cruz, USA), PU.1 (sc352-Santa Cruz, USA), DNMT1 (Ab13537-Abcam, UK), H3K9Ac (07-352-Upstate, USA), H3K9Me3 (Ab88-98-Abcam, UK), H3K4Me3 (pAb003-050-Diagenode, Belgium), H3K27Me3 (Ab6002-Abcam, UK), and control antibody (NI01-EMB Biosciences, USA).

    Techniques: Over Expression, Chromatin Immunoprecipitation, Transfection

    Chromatin state discovery and characterization a, Top: Profiles for nine chromatin marks (grayscale) are shown across the wntless (WLS) gene in four cell types, and summarized in a single chromatin state annotation track for each (colored according to b ). WLS is poised in ES cells, repressed in GM12878 cells, and transcribed in HUVEC and NHLF. Its TSS switches accordingly between poised (purple), repressed (grey) and active (red) promoter states; enhancer regions within the gene body become strongly activated (orange, yellow); and its gene body changes from low signal (white) to transcribed (green). These chromatin state changes summarize coordinated changes in many chromatin marks; for example, H3K27me3, H3K4me3 and H3K4me2 jointly mark a poised promoter, while loss of H3K27me3 and gain of H3K27ac and H3K9ac mark promoter activation. Bottom: Nine chromatin state tracks, one per cell type, in a 900kb region centered at WLS summarize 90 chromatin tracks in directly-interpretable dynamic annotations, showing activation and repression patterns for 6 genes and hundreds of regulatory regions, including enhancer states. b, Chromatin states learned jointly across cell types by a multivariate HMM. Table shows emission parameters learned de novo based on genome-wide recurrent combinations of chromatin marks. Each entry denotes the frequency with which a given mark is found at genomic positions corresponding to the chromatin state. c, Genome coverage, functional enrichments, and candidate annotations for each chromatin state. Blue shading indicates intensity, scaled by column. d, Box plot depicts enhancer activity for predicted regulatory elements. 250bp-long sequences corresponding to strong or weak/poised HepG2 enhancer elements, or GM12878-specific strong enhancer elements were inserted upstream of a luciferase gene and transfected into HepG2 cells. Reporter activity was measured in relative light units. Robust activity is seen for strong enhancers in the matched cell type, but not for weak/poised enhancers or for strong enhancers specific to a different cell type. Box-and-whiskers indicate 5 th , 25 th , 50 th , 75 th and 95 th percentiles.

    Journal: Nature

    Article Title: Systematic analysis of chromatin state dynamics in nine human cell types

    doi: 10.1038/nature09906

    Figure Lengend Snippet: Chromatin state discovery and characterization a, Top: Profiles for nine chromatin marks (grayscale) are shown across the wntless (WLS) gene in four cell types, and summarized in a single chromatin state annotation track for each (colored according to b ). WLS is poised in ES cells, repressed in GM12878 cells, and transcribed in HUVEC and NHLF. Its TSS switches accordingly between poised (purple), repressed (grey) and active (red) promoter states; enhancer regions within the gene body become strongly activated (orange, yellow); and its gene body changes from low signal (white) to transcribed (green). These chromatin state changes summarize coordinated changes in many chromatin marks; for example, H3K27me3, H3K4me3 and H3K4me2 jointly mark a poised promoter, while loss of H3K27me3 and gain of H3K27ac and H3K9ac mark promoter activation. Bottom: Nine chromatin state tracks, one per cell type, in a 900kb region centered at WLS summarize 90 chromatin tracks in directly-interpretable dynamic annotations, showing activation and repression patterns for 6 genes and hundreds of regulatory regions, including enhancer states. b, Chromatin states learned jointly across cell types by a multivariate HMM. Table shows emission parameters learned de novo based on genome-wide recurrent combinations of chromatin marks. Each entry denotes the frequency with which a given mark is found at genomic positions corresponding to the chromatin state. c, Genome coverage, functional enrichments, and candidate annotations for each chromatin state. Blue shading indicates intensity, scaled by column. d, Box plot depicts enhancer activity for predicted regulatory elements. 250bp-long sequences corresponding to strong or weak/poised HepG2 enhancer elements, or GM12878-specific strong enhancer elements were inserted upstream of a luciferase gene and transfected into HepG2 cells. Reporter activity was measured in relative light units. Robust activity is seen for strong enhancers in the matched cell type, but not for weak/poised enhancers or for strong enhancers specific to a different cell type. Box-and-whiskers indicate 5 th , 25 th , 50 th , 75 th and 95 th percentiles.

    Article Snippet: Antibodies ChIP assays were performed using the following antibody reagents: H3K4me1 (Abcam ab8895, lot 38311/659352), H3K4me2 (Abcam ab7766, lot 56293), H3K4me3 (Abcam ab8580, lot 331024; Milipore 04-473, lot DAM1623866), H3K9ac (Abcam ab44441, lot 455103/550799), H3K27ac (Abcam ab4729, lot 31456), H3K36me3 (Abcam ab9050, lot 136353), H4K20me1 (Abcam ab9051, lot 104513/519198), H3K27me3 (Millipore 07-449, lot DAM1387952/DAM1514011), CTCF (Millipore 07-729, lot 1350637), H3K9me3 (Abcam ab8898, lot 484088), H2A.Z (Millipore 07-594, lot DAM1504736) and RNAPII N-terminus (Santa Cruz sc-899X, lot H0510).

    Techniques: Activation Assay, Genome Wide, Functional Assay, Activity Assay, Luciferase, Transfection

    Anti-H3K64ac antibody validation. ( A ) Western blot analysis with H3K64ac antibody (left panel) on nuclear extracts from untreated (−) or Na-butyrate-treated (+) HeLa cells. Ponceau staining is shown as loading control (right panel). ( B ), ( C ), ( D ), and ( E ) Immuno-dot blots showing specific reactivity of the H3K64ac antibody. Aliquots of indicated pmoles of linear H3 peptides were spotted on the membrane. Of note, detection of peptides in ( E ) has been checked with H3K9ac, H3K18ac, and H3K27ac specific antibodies. ( F ) Peptide competition assay of H3K64ac immunoblot. H3K64ac antibody was pre-adsorbed with 50 pmoles/ml of indicated peptides. Ponceau staining is shown as loading control (bottom panel). ( G ) The H3K64ac antibody detects globular, tailless H3. Nucleosomes from NIH3T3 cells were incubated with no (−) or increasing amounts of trypsin to cleave the histone tails and immunoblotted as indicated for H3K9ac, H3K18ac, H3K27ac, H3K64ac and H3. The position of full-length and tailless H3 is indicated and loading was controlled by ponceau staining. ( H ) Peptide competition assay in immunofluorescence. MEFs were treated overnight with Na-butyrate (NaB) and then stained with H3K64ac antibody, pre-incubated with 100 pmoles/ml of either unmodified or acetylated K64 peptide. ( I ) Immunoblot analysis of H3K64 acetylation state in additional cell lines (Drosophila S2, mouse MEFs, human HeLa) with H3K64ac antibody. Inhibition of HDACs by Na-butyrate treatment increases acetylation levels. Ponceau staining is shown as loading control (bottom panel). DOI: http://dx.doi.org/10.7554/eLife.01632.005

    Journal: eLife

    Article Title: Acetylation of histone H3 at lysine 64 regulates nucleosome dynamics and facilitates transcription

    doi: 10.7554/eLife.01632

    Figure Lengend Snippet: Anti-H3K64ac antibody validation. ( A ) Western blot analysis with H3K64ac antibody (left panel) on nuclear extracts from untreated (−) or Na-butyrate-treated (+) HeLa cells. Ponceau staining is shown as loading control (right panel). ( B ), ( C ), ( D ), and ( E ) Immuno-dot blots showing specific reactivity of the H3K64ac antibody. Aliquots of indicated pmoles of linear H3 peptides were spotted on the membrane. Of note, detection of peptides in ( E ) has been checked with H3K9ac, H3K18ac, and H3K27ac specific antibodies. ( F ) Peptide competition assay of H3K64ac immunoblot. H3K64ac antibody was pre-adsorbed with 50 pmoles/ml of indicated peptides. Ponceau staining is shown as loading control (bottom panel). ( G ) The H3K64ac antibody detects globular, tailless H3. Nucleosomes from NIH3T3 cells were incubated with no (−) or increasing amounts of trypsin to cleave the histone tails and immunoblotted as indicated for H3K9ac, H3K18ac, H3K27ac, H3K64ac and H3. The position of full-length and tailless H3 is indicated and loading was controlled by ponceau staining. ( H ) Peptide competition assay in immunofluorescence. MEFs were treated overnight with Na-butyrate (NaB) and then stained with H3K64ac antibody, pre-incubated with 100 pmoles/ml of either unmodified or acetylated K64 peptide. ( I ) Immunoblot analysis of H3K64 acetylation state in additional cell lines (Drosophila S2, mouse MEFs, human HeLa) with H3K64ac antibody. Inhibition of HDACs by Na-butyrate treatment increases acetylation levels. Ponceau staining is shown as loading control (bottom panel). DOI: http://dx.doi.org/10.7554/eLife.01632.005

    Article Snippet: Antibodies Histone modifications primary antibodies: H3K4me3 (Millipore, Billerica, MA), H3K9ac (Cell Signaling, Boston, MA), ChIP grade H3K9ac (Abcam, UK), H3K9ac (Millipore, Billerica, MA), H3K9me3 (Millipore), H3K14ac (Millipore), H3K18ac (Cell Signaling), H3K18ac (Abcam), ChIP grade H3K27ac (Abcam), H3K27me3 (Millipore), H3 (Abcam), H4K16ac (Santa Cruz Biotechnology Inc., Dallas, Texas).

    Techniques: Western Blot, Staining, Competitive Binding Assay, Incubation, Immunofluorescence, Inhibition

    Validation of the substrates used in the in vitro assays. ( A ) Validation of H3 acetylation on the octamer substrates used in the in vitro assays. The immunoblots show the acetylation levels of the reconstituted octamers specifically acetylated either on H3K64 or H3K9 compared with the unmodified H3. Ponceau staining is shown as loading control. ( B ) Staining of protein gel used to check that equal amounts of proteins had been used in the experiments and to assess H2A–H2B dimer, as well as unmodified, H3K64ac, and H3K9ac tetramer qualities. DOI: http://dx.doi.org/10.7554/eLife.01632.016

    Journal: eLife

    Article Title: Acetylation of histone H3 at lysine 64 regulates nucleosome dynamics and facilitates transcription

    doi: 10.7554/eLife.01632

    Figure Lengend Snippet: Validation of the substrates used in the in vitro assays. ( A ) Validation of H3 acetylation on the octamer substrates used in the in vitro assays. The immunoblots show the acetylation levels of the reconstituted octamers specifically acetylated either on H3K64 or H3K9 compared with the unmodified H3. Ponceau staining is shown as loading control. ( B ) Staining of protein gel used to check that equal amounts of proteins had been used in the experiments and to assess H2A–H2B dimer, as well as unmodified, H3K64ac, and H3K9ac tetramer qualities. DOI: http://dx.doi.org/10.7554/eLife.01632.016

    Article Snippet: Antibodies Histone modifications primary antibodies: H3K4me3 (Millipore, Billerica, MA), H3K9ac (Cell Signaling, Boston, MA), ChIP grade H3K9ac (Abcam, UK), H3K9ac (Millipore, Billerica, MA), H3K9me3 (Millipore), H3K14ac (Millipore), H3K18ac (Cell Signaling), H3K18ac (Abcam), ChIP grade H3K27ac (Abcam), H3K27me3 (Millipore), H3 (Abcam), H4K16ac (Santa Cruz Biotechnology Inc., Dallas, Texas).

    Techniques: In Vitro, Western Blot, Staining

    H3K64ac distribution within active chromatin. ( A ) Scatterplot showing global correlation between raw data from the two biological H3K64ac ChIP-on-chip replicate experiments. The green line is a loess-fitted trend line. ( B ) UCSC browser screenshots of H3K64ac enrichment at the proximal promoter of active genes ( Eif3a , upper panel and Taf5 , lower panel). ( C ) UCSC browser screenshots of H3K64ac enrichment at the proximal promoter of inactive genes ( Cyp2c55 and Cyp2c65 ). For ( B ) and ( C ) shown are the enrichments for H3K64ac (as log2 values of ChIP/input) for one biological replicate. Areas in light blue highlight the region surrounding the TSS of the indicated genes. ( D ) Scatterplots showing global correlation at TSS between H3K64ac and ‘active’ modifications such as H3K9ac and H3K4me2 and ‘repressive’ marks such as H3K27me3 and H3K64me3. The green line is a loess-fitted trend line. ( E ) Meta-gene plot showing total H3 enrichment around TSS grouped according to their expression level. Of note, H3 density is rather uniform and the slight enrichment proximal to the TSS cannot explain the strong enrichment of H3K64ac detected in the same region ( Figure 2C ). DOI: http://dx.doi.org/10.7554/eLife.01632.008

    Journal: eLife

    Article Title: Acetylation of histone H3 at lysine 64 regulates nucleosome dynamics and facilitates transcription

    doi: 10.7554/eLife.01632

    Figure Lengend Snippet: H3K64ac distribution within active chromatin. ( A ) Scatterplot showing global correlation between raw data from the two biological H3K64ac ChIP-on-chip replicate experiments. The green line is a loess-fitted trend line. ( B ) UCSC browser screenshots of H3K64ac enrichment at the proximal promoter of active genes ( Eif3a , upper panel and Taf5 , lower panel). ( C ) UCSC browser screenshots of H3K64ac enrichment at the proximal promoter of inactive genes ( Cyp2c55 and Cyp2c65 ). For ( B ) and ( C ) shown are the enrichments for H3K64ac (as log2 values of ChIP/input) for one biological replicate. Areas in light blue highlight the region surrounding the TSS of the indicated genes. ( D ) Scatterplots showing global correlation at TSS between H3K64ac and ‘active’ modifications such as H3K9ac and H3K4me2 and ‘repressive’ marks such as H3K27me3 and H3K64me3. The green line is a loess-fitted trend line. ( E ) Meta-gene plot showing total H3 enrichment around TSS grouped according to their expression level. Of note, H3 density is rather uniform and the slight enrichment proximal to the TSS cannot explain the strong enrichment of H3K64ac detected in the same region ( Figure 2C ). DOI: http://dx.doi.org/10.7554/eLife.01632.008

    Article Snippet: Antibodies Histone modifications primary antibodies: H3K4me3 (Millipore, Billerica, MA), H3K9ac (Cell Signaling, Boston, MA), ChIP grade H3K9ac (Abcam, UK), H3K9ac (Millipore, Billerica, MA), H3K9me3 (Millipore), H3K14ac (Millipore), H3K18ac (Cell Signaling), H3K18ac (Abcam), ChIP grade H3K27ac (Abcam), H3K27me3 (Millipore), H3 (Abcam), H4K16ac (Santa Cruz Biotechnology Inc., Dallas, Texas).

    Techniques: Chromatin Immunoprecipitation, Expressing

    (A ) Western blot of total protein samples of Ada2b d842 mutant, w 1118 control, and Ada2bEGFP transgene carrier Ada2b d842 animals. The labels are P: pupa, L: larvae Ada2b resc : Ada2bEGFP transgene carrier Ada2b d842 and as indicated. The Mw of the two dADA2b isoforms are indicated. Note that in Ada2b resc the EGFP tag attached to the C-terminus of the larger dADA2b isoform increases its size. At the bottom on the left panel the same filter developed with alpha-tubulin-specific Ab as loading control is shown. ( B ) Western blot of total protein samples of Ada2b d842 mutants, w 1118 control and Ada2bEGFP transgene carrier Ada2b d842 animals developed with histone H3K9ac-specific antibody. Labels, genotypes and developmental stages are as indicate, and as in (A). On the bottom: the same filters developed with anti-histone H3 antibody. ( C ) Immunostaining of salivary glands (top) and of polytene chromosomes (bottom) of late third instar Ada2b d842 , control ( w 1118 ) and Ada2bEGFP transgene carrier Ada2b d842 larvae with H3K9ac- and H3K14ac-specific Abs. Pol II-specific staining (Ab: 7G5) of the same polytene chromosomes is shown as staining controls. ( D ) Accumulation of H3K9ac is not detectable in wild type ( w 1118 ) heat-shock puff (top). Puff is formed in the absence of H3K9ac in Ada2b d842 mutant (bottom). Puffs formed at the 93D cytological region are indicated by arrow as an example. Red: H3K9ac-specific, green: Pol II-specific Ab (H14) staining.

    Journal: Nucleic Acids Research

    Article Title: The loss of histone H3 lysine 9 acetylation due to dSAGA-specific dAda2b mutation influences the expression of only a small subset of genes

    doi: 10.1093/nar/gkp722

    Figure Lengend Snippet: (A ) Western blot of total protein samples of Ada2b d842 mutant, w 1118 control, and Ada2bEGFP transgene carrier Ada2b d842 animals. The labels are P: pupa, L: larvae Ada2b resc : Ada2bEGFP transgene carrier Ada2b d842 and as indicated. The Mw of the two dADA2b isoforms are indicated. Note that in Ada2b resc the EGFP tag attached to the C-terminus of the larger dADA2b isoform increases its size. At the bottom on the left panel the same filter developed with alpha-tubulin-specific Ab as loading control is shown. ( B ) Western blot of total protein samples of Ada2b d842 mutants, w 1118 control and Ada2bEGFP transgene carrier Ada2b d842 animals developed with histone H3K9ac-specific antibody. Labels, genotypes and developmental stages are as indicate, and as in (A). On the bottom: the same filters developed with anti-histone H3 antibody. ( C ) Immunostaining of salivary glands (top) and of polytene chromosomes (bottom) of late third instar Ada2b d842 , control ( w 1118 ) and Ada2bEGFP transgene carrier Ada2b d842 larvae with H3K9ac- and H3K14ac-specific Abs. Pol II-specific staining (Ab: 7G5) of the same polytene chromosomes is shown as staining controls. ( D ) Accumulation of H3K9ac is not detectable in wild type ( w 1118 ) heat-shock puff (top). Puff is formed in the absence of H3K9ac in Ada2b d842 mutant (bottom). Puffs formed at the 93D cytological region are indicated by arrow as an example. Red: H3K9ac-specific, green: Pol II-specific Ab (H14) staining.

    Article Snippet: Treatment with anti-H3K9ac primary antibody (1 : 200, Abcam) at 4°C was followed by Alexa Fluor 555-conjugated anti-rabbit secondary antibody (Molecular Probes).

    Techniques: Western Blot, Mutagenesis, Immunostaining, Staining

    ( A ) Changes in the levels of specific mRNAs in dAda2b d842 mutants compared to wild type ( w 1118 ) samples. Average changes in mRNA levels determined by microarray hybridizations in three biological samples are shown. ( B–E) Q-PCR detection of specific fragments of selected genes in chromatin immunoprecipitated samples. The primers used for Q-PCR are listed in Table 1 . H3K9ac-specific (B and C), dADAb- (D) and Pol II-specific (E) antibody-precipitated chromatin from wild type ( w 1118 ) and dAda2b samples obtained from sycronised third instar larvae. C t and dCt values of representative experiments of those shown here are given in Table 3 . Note that ChIP experiments to detect H3K9ac in the intergenic regions (C right) were done in separate experiments from those shown on B left.

    Journal: Nucleic Acids Research

    Article Title: The loss of histone H3 lysine 9 acetylation due to dSAGA-specific dAda2b mutation influences the expression of only a small subset of genes

    doi: 10.1093/nar/gkp722

    Figure Lengend Snippet: ( A ) Changes in the levels of specific mRNAs in dAda2b d842 mutants compared to wild type ( w 1118 ) samples. Average changes in mRNA levels determined by microarray hybridizations in three biological samples are shown. ( B–E) Q-PCR detection of specific fragments of selected genes in chromatin immunoprecipitated samples. The primers used for Q-PCR are listed in Table 1 . H3K9ac-specific (B and C), dADAb- (D) and Pol II-specific (E) antibody-precipitated chromatin from wild type ( w 1118 ) and dAda2b samples obtained from sycronised third instar larvae. C t and dCt values of representative experiments of those shown here are given in Table 3 . Note that ChIP experiments to detect H3K9ac in the intergenic regions (C right) were done in separate experiments from those shown on B left.

    Article Snippet: Treatment with anti-H3K9ac primary antibody (1 : 200, Abcam) at 4°C was followed by Alexa Fluor 555-conjugated anti-rabbit secondary antibody (Molecular Probes).

    Techniques: Microarray, Polymerase Chain Reaction, Immunoprecipitation, Chromatin Immunoprecipitation

    The histone 3 lysine 9 monomethylation (H3K9me1) level was analyzed as the mean gray value of hMSCs cultured on the MARC chip for 7 days. All data shown as average ± SD. Comparison of experimental replicas (N = 4) with at least 100 cells were analyzed in each of the experimental replica on each pattern type. * p represents statistical significance with p ≤ 0.05, ** p ≤ 0.01. Refer to Table 1 for abbreviations.

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Temporal Changes in Nucleus Morphology, Lamin A/C and Histone Methylation During Nanotopography-Induced Neuronal Differentiation of Stem Cells

    doi: 10.3389/fbioe.2018.00069

    Figure Lengend Snippet: The histone 3 lysine 9 monomethylation (H3K9me1) level was analyzed as the mean gray value of hMSCs cultured on the MARC chip for 7 days. All data shown as average ± SD. Comparison of experimental replicas (N = 4) with at least 100 cells were analyzed in each of the experimental replica on each pattern type. * p represents statistical significance with p ≤ 0.05, ** p ≤ 0.01. Refer to Table 1 for abbreviations.

    Article Snippet: Then for hESCs, samples were incubated with mouse anti-lamin A/C (1:100), rabbit anti-histone H3 trimethyl K9 (H3K9me3, 1:200, Abcam), or mouse anti- histone 3 acetylation on Lysine 9 (H3K9ac, 1:500, Abcam).

    Techniques: Cell Culture, Chromatin Immunoprecipitation

    (A ) Western blot of total protein samples of Ada2b d842 mutant, w 1118 control, and Ada2bEGFP transgene carrier Ada2b d842 animals. The labels are P: pupa, L: larvae Ada2b resc : Ada2bEGFP transgene carrier Ada2b d842 and as indicated. The Mw of the two dADA2b isoforms are indicated. Note that in Ada2b resc the EGFP tag attached to the C-terminus of the larger dADA2b isoform increases its size. At the bottom on the left panel the same filter developed with alpha-tubulin-specific Ab as loading control is shown. ( B ) Western blot of total protein samples of Ada2b d842 mutants, w 1118 control and Ada2bEGFP transgene carrier Ada2b d842 animals developed with histone H3K9ac-specific antibody. Labels, genotypes and developmental stages are as indicate, and as in (A). On the bottom: the same filters developed with anti-histone H3 antibody. ( C ) Immunostaining of salivary glands (top) and of polytene chromosomes (bottom) of late third instar Ada2b d842 , control ( w 1118 ) and Ada2bEGFP transgene carrier Ada2b d842 larvae with H3K9ac- and H3K14ac-specific Abs. Pol II-specific staining (Ab: 7G5) of the same polytene chromosomes is shown as staining controls. ( D ) Accumulation of H3K9ac is not detectable in wild type ( w 1118 ) heat-shock puff (top). Puff is formed in the absence of H3K9ac in Ada2b d842 mutant (bottom). Puffs formed at the 93D cytological region are indicated by arrow as an example. Red: H3K9ac-specific, green: Pol II-specific Ab (H14) staining.

    Journal: Nucleic Acids Research

    Article Title: The loss of histone H3 lysine 9 acetylation due to dSAGA-specific dAda2b mutation influences the expression of only a small subset of genes

    doi: 10.1093/nar/gkp722

    Figure Lengend Snippet: (A ) Western blot of total protein samples of Ada2b d842 mutant, w 1118 control, and Ada2bEGFP transgene carrier Ada2b d842 animals. The labels are P: pupa, L: larvae Ada2b resc : Ada2bEGFP transgene carrier Ada2b d842 and as indicated. The Mw of the two dADA2b isoforms are indicated. Note that in Ada2b resc the EGFP tag attached to the C-terminus of the larger dADA2b isoform increases its size. At the bottom on the left panel the same filter developed with alpha-tubulin-specific Ab as loading control is shown. ( B ) Western blot of total protein samples of Ada2b d842 mutants, w 1118 control and Ada2bEGFP transgene carrier Ada2b d842 animals developed with histone H3K9ac-specific antibody. Labels, genotypes and developmental stages are as indicate, and as in (A). On the bottom: the same filters developed with anti-histone H3 antibody. ( C ) Immunostaining of salivary glands (top) and of polytene chromosomes (bottom) of late third instar Ada2b d842 , control ( w 1118 ) and Ada2bEGFP transgene carrier Ada2b d842 larvae with H3K9ac- and H3K14ac-specific Abs. Pol II-specific staining (Ab: 7G5) of the same polytene chromosomes is shown as staining controls. ( D ) Accumulation of H3K9ac is not detectable in wild type ( w 1118 ) heat-shock puff (top). Puff is formed in the absence of H3K9ac in Ada2b d842 mutant (bottom). Puffs formed at the 93D cytological region are indicated by arrow as an example. Red: H3K9ac-specific, green: Pol II-specific Ab (H14) staining.

    Article Snippet: H3K9ac-specific antibody was from Abcam (dilution 1 : 200), H3K14ac-specific antibody was from Upstate (07-353, dilution 1 : 200), Pol II specific antibodies were 7G5 ( ) or H14 (Covance Research Products) (dilution 1 : 500) as indicated.

    Techniques: Western Blot, Mutagenesis, Immunostaining, Staining

    ( A ) Changes in the levels of specific mRNAs in dAda2b d842 mutants compared to wild type ( w 1118 ) samples. Average changes in mRNA levels determined by microarray hybridizations in three biological samples are shown. ( B–E) Q-PCR detection of specific fragments of selected genes in chromatin immunoprecipitated samples. The primers used for Q-PCR are listed in Table 1 . H3K9ac-specific (B and C), dADAb- (D) and Pol II-specific (E) antibody-precipitated chromatin from wild type ( w 1118 ) and dAda2b samples obtained from sycronised third instar larvae. C t and dCt values of representative experiments of those shown here are given in Table 3 . Note that ChIP experiments to detect H3K9ac in the intergenic regions (C right) were done in separate experiments from those shown on B left.

    Journal: Nucleic Acids Research

    Article Title: The loss of histone H3 lysine 9 acetylation due to dSAGA-specific dAda2b mutation influences the expression of only a small subset of genes

    doi: 10.1093/nar/gkp722

    Figure Lengend Snippet: ( A ) Changes in the levels of specific mRNAs in dAda2b d842 mutants compared to wild type ( w 1118 ) samples. Average changes in mRNA levels determined by microarray hybridizations in three biological samples are shown. ( B–E) Q-PCR detection of specific fragments of selected genes in chromatin immunoprecipitated samples. The primers used for Q-PCR are listed in Table 1 . H3K9ac-specific (B and C), dADAb- (D) and Pol II-specific (E) antibody-precipitated chromatin from wild type ( w 1118 ) and dAda2b samples obtained from sycronised third instar larvae. C t and dCt values of representative experiments of those shown here are given in Table 3 . Note that ChIP experiments to detect H3K9ac in the intergenic regions (C right) were done in separate experiments from those shown on B left.

    Article Snippet: H3K9ac-specific antibody was from Abcam (dilution 1 : 200), H3K14ac-specific antibody was from Upstate (07-353, dilution 1 : 200), Pol II specific antibodies were 7G5 ( ) or H14 (Covance Research Products) (dilution 1 : 500) as indicated.

    Techniques: Microarray, Polymerase Chain Reaction, Immunoprecipitation, Chromatin Immunoprecipitation

    (A ) Western blot of total protein samples of Ada2b d842 mutant, w 1118 control, and Ada2bEGFP transgene carrier Ada2b d842 animals. The labels are P: pupa, L: larvae Ada2b resc : Ada2bEGFP transgene carrier Ada2b d842 and as indicated. The Mw of the two dADA2b isoforms are indicated. Note that in Ada2b resc the EGFP tag attached to the C-terminus of the larger dADA2b isoform increases its size. At the bottom on the left panel the same filter developed with alpha-tubulin-specific Ab as loading control is shown. ( B ) Western blot of total protein samples of Ada2b d842 mutants, w 1118 control and Ada2bEGFP transgene carrier Ada2b d842 animals developed with histone H3K9ac-specific antibody. Labels, genotypes and developmental stages are as indicate, and as in (A). On the bottom: the same filters developed with anti-histone H3 antibody. ( C ) Immunostaining of salivary glands (top) and of polytene chromosomes (bottom) of late third instar Ada2b d842 , control ( w 1118 ) and Ada2bEGFP transgene carrier Ada2b d842 larvae with H3K9ac- and H3K14ac-specific Abs. Pol II-specific staining (Ab: 7G5) of the same polytene chromosomes is shown as staining controls. ( D ) Accumulation of H3K9ac is not detectable in wild type ( w 1118 ) heat-shock puff (top). Puff is formed in the absence of H3K9ac in Ada2b d842 mutant (bottom). Puffs formed at the 93D cytological region are indicated by arrow as an example. Red: H3K9ac-specific, green: Pol II-specific Ab (H14) staining.

    Journal: Nucleic Acids Research

    Article Title: The loss of histone H3 lysine 9 acetylation due to dSAGA-specific dAda2b mutation influences the expression of only a small subset of genes

    doi: 10.1093/nar/gkp722

    Figure Lengend Snippet: (A ) Western blot of total protein samples of Ada2b d842 mutant, w 1118 control, and Ada2bEGFP transgene carrier Ada2b d842 animals. The labels are P: pupa, L: larvae Ada2b resc : Ada2bEGFP transgene carrier Ada2b d842 and as indicated. The Mw of the two dADA2b isoforms are indicated. Note that in Ada2b resc the EGFP tag attached to the C-terminus of the larger dADA2b isoform increases its size. At the bottom on the left panel the same filter developed with alpha-tubulin-specific Ab as loading control is shown. ( B ) Western blot of total protein samples of Ada2b d842 mutants, w 1118 control and Ada2bEGFP transgene carrier Ada2b d842 animals developed with histone H3K9ac-specific antibody. Labels, genotypes and developmental stages are as indicate, and as in (A). On the bottom: the same filters developed with anti-histone H3 antibody. ( C ) Immunostaining of salivary glands (top) and of polytene chromosomes (bottom) of late third instar Ada2b d842 , control ( w 1118 ) and Ada2bEGFP transgene carrier Ada2b d842 larvae with H3K9ac- and H3K14ac-specific Abs. Pol II-specific staining (Ab: 7G5) of the same polytene chromosomes is shown as staining controls. ( D ) Accumulation of H3K9ac is not detectable in wild type ( w 1118 ) heat-shock puff (top). Puff is formed in the absence of H3K9ac in Ada2b d842 mutant (bottom). Puffs formed at the 93D cytological region are indicated by arrow as an example. Red: H3K9ac-specific, green: Pol II-specific Ab (H14) staining.

    Article Snippet: dAda2b mutation results in a drastic decrease in histone H3K9ac and H3K14ac levels Recently, we reported the isolation of a dAda2b null allele (dAda2bd842 ) and showed that the loss of dAda2b function results in lethality in later developmental stages, and a decrease of histone H3K9ac and H3K14ac levels on polytene chromosomes ( ).

    Techniques: Western Blot, Mutagenesis, Immunostaining, Staining

    ( A ) Changes in the levels of specific mRNAs in dAda2b d842 mutants compared to wild type ( w 1118 ) samples. Average changes in mRNA levels determined by microarray hybridizations in three biological samples are shown. ( B–E) Q-PCR detection of specific fragments of selected genes in chromatin immunoprecipitated samples. The primers used for Q-PCR are listed in Table 1 . H3K9ac-specific (B and C), dADAb- (D) and Pol II-specific (E) antibody-precipitated chromatin from wild type ( w 1118 ) and dAda2b samples obtained from sycronised third instar larvae. C t and dCt values of representative experiments of those shown here are given in Table 3 . Note that ChIP experiments to detect H3K9ac in the intergenic regions (C right) were done in separate experiments from those shown on B left.

    Journal: Nucleic Acids Research

    Article Title: The loss of histone H3 lysine 9 acetylation due to dSAGA-specific dAda2b mutation influences the expression of only a small subset of genes

    doi: 10.1093/nar/gkp722

    Figure Lengend Snippet: ( A ) Changes in the levels of specific mRNAs in dAda2b d842 mutants compared to wild type ( w 1118 ) samples. Average changes in mRNA levels determined by microarray hybridizations in three biological samples are shown. ( B–E) Q-PCR detection of specific fragments of selected genes in chromatin immunoprecipitated samples. The primers used for Q-PCR are listed in Table 1 . H3K9ac-specific (B and C), dADAb- (D) and Pol II-specific (E) antibody-precipitated chromatin from wild type ( w 1118 ) and dAda2b samples obtained from sycronised third instar larvae. C t and dCt values of representative experiments of those shown here are given in Table 3 . Note that ChIP experiments to detect H3K9ac in the intergenic regions (C right) were done in separate experiments from those shown on B left.

    Article Snippet: dAda2b mutation results in a drastic decrease in histone H3K9ac and H3K14ac levels Recently, we reported the isolation of a dAda2b null allele (dAda2bd842 ) and showed that the loss of dAda2b function results in lethality in later developmental stages, and a decrease of histone H3K9ac and H3K14ac levels on polytene chromosomes ( ).

    Techniques: Microarray, Polymerase Chain Reaction, Immunoprecipitation, Chromatin Immunoprecipitation

    Identification of positively stained and negative tumour cells using the Ariol system. The Ariol system trainer overlay shows correct identification of positive (yellow dots) and negative (blue dots) cells on tumour cores. TMA slides were scanned using a × 20 magnification. Shown for both H3K9Ac and H3K27me3 are highly positive tumour cores ( A and D ), tumour cores with both positive and negative cells ( B and E ) and negative tumour cores ( C and F ). The Ariol system was trained to identify positive and negative cells for each individual marker.

    Journal: British Journal of Cancer

    Article Title: Epigenetic status of LINE-1 predicts clinical outcome in early-stage rectal cancer

    doi: 10.1038/bjc.2013.654

    Figure Lengend Snippet: Identification of positively stained and negative tumour cells using the Ariol system. The Ariol system trainer overlay shows correct identification of positive (yellow dots) and negative (blue dots) cells on tumour cores. TMA slides were scanned using a × 20 magnification. Shown for both H3K9Ac and H3K27me3 are highly positive tumour cores ( A and D ), tumour cores with both positive and negative cells ( B and E ) and negative tumour cores ( C and F ). The Ariol system was trained to identify positive and negative cells for each individual marker.

    Article Snippet: TMA sections were stained using mouse anti-H3K27me3 (dilution 1 : 200; ab6002, Abcam, Cambridge, UK) or rabbit anti-H3K9Ac (dilution 1 : 600, ab8898, Abcam), using a standard IHC protocol ( ).

    Techniques: Staining, Marker

    Kaplan–Meier curves of histone modifications H3K27me3 and H3K9Ac by expression levels. Kaplan–Meier curves were made to visualise differences in survival and recurrence between patients with high and low expression of H3K27me3. Log-rank tests were performed to calculate the difference between the low and high H3K27me3 expression groups. Survival times were calculated as time from surgery till an event (death or recurrence). ( A ) Kaplan–Meier curves showing overall survival of the high and low methylation groups based on H3K27me3 expression. ( B ) Kaplan–Meier curves showing overall survival in the high and low methylation groups based on H3K9Ac expression.

    Journal: British Journal of Cancer

    Article Title: Epigenetic status of LINE-1 predicts clinical outcome in early-stage rectal cancer

    doi: 10.1038/bjc.2013.654

    Figure Lengend Snippet: Kaplan–Meier curves of histone modifications H3K27me3 and H3K9Ac by expression levels. Kaplan–Meier curves were made to visualise differences in survival and recurrence between patients with high and low expression of H3K27me3. Log-rank tests were performed to calculate the difference between the low and high H3K27me3 expression groups. Survival times were calculated as time from surgery till an event (death or recurrence). ( A ) Kaplan–Meier curves showing overall survival of the high and low methylation groups based on H3K27me3 expression. ( B ) Kaplan–Meier curves showing overall survival in the high and low methylation groups based on H3K9Ac expression.

    Article Snippet: TMA sections were stained using mouse anti-H3K27me3 (dilution 1 : 200; ab6002, Abcam, Cambridge, UK) or rabbit anti-H3K9Ac (dilution 1 : 600, ab8898, Abcam), using a standard IHC protocol ( ).

    Techniques: Expressing, Methylation

    Kaplan–Meier curves of histone modifications H3K27me3 and H3K9Ac according to LINE-1 or Alu methylation levels. Kaplan–Meier curves were made to visualise differences in survival and recurrence between patients with high and low expression of H3K27me3 and H3K9Ac. Log-rank tests were performed to calculate the difference between the low and high H3K27me3 or H3K9Ac expression groups, respectively. Survival times were calculated as time from surgery till an event (death or recurrence). ( A ) Overall survival based on H3K27me3 expression in the LINE-1 methylation low group. ( B ) Overall survival based on H3K27me3 expression in the Alu methylation low group. ( C ) Local recurrence-free survival based on H3K27me3 expression in the LINE-1 methylation low group. ( D ) Local recurrence-free survival based on H3K27me3 expression in the Alu methylation low group. ( E ) Local recurrence-free survival based on H3K9Ac expression in the LINE-1 methylation high group. ( F ) Local recurrence-free survival based on H3K9Ac expression in the Alu methylation high group.

    Journal: British Journal of Cancer

    Article Title: Epigenetic status of LINE-1 predicts clinical outcome in early-stage rectal cancer

    doi: 10.1038/bjc.2013.654

    Figure Lengend Snippet: Kaplan–Meier curves of histone modifications H3K27me3 and H3K9Ac according to LINE-1 or Alu methylation levels. Kaplan–Meier curves were made to visualise differences in survival and recurrence between patients with high and low expression of H3K27me3 and H3K9Ac. Log-rank tests were performed to calculate the difference between the low and high H3K27me3 or H3K9Ac expression groups, respectively. Survival times were calculated as time from surgery till an event (death or recurrence). ( A ) Overall survival based on H3K27me3 expression in the LINE-1 methylation low group. ( B ) Overall survival based on H3K27me3 expression in the Alu methylation low group. ( C ) Local recurrence-free survival based on H3K27me3 expression in the LINE-1 methylation low group. ( D ) Local recurrence-free survival based on H3K27me3 expression in the Alu methylation low group. ( E ) Local recurrence-free survival based on H3K9Ac expression in the LINE-1 methylation high group. ( F ) Local recurrence-free survival based on H3K9Ac expression in the Alu methylation high group.

    Article Snippet: TMA sections were stained using mouse anti-H3K27me3 (dilution 1 : 200; ab6002, Abcam, Cambridge, UK) or rabbit anti-H3K9Ac (dilution 1 : 600, ab8898, Abcam), using a standard IHC protocol ( ).

    Techniques: Methylation, Expressing

    MPA treatment increases global histone acetylation in Caco-2 cells . (A) Dot-blot analysis; Caco-2 cells were cultured in six-well plates and post confluence monolayers were treated either with 10 μM MPA or DMSO for indicated time points. Total proteins were extracted and a volume of 2 μl from each time point was spotted on nitrocellulose membrane, dried and then probed with anti-H3K9ac and anti-H4K8ac antibodies. (B) Western blot analysis; Total cell proteins were resolved on 12.5% SDS-PAGE gels and, immunoblotted using anti-H3K9ac and anti-H4K8ac specific antibodies. β-actin was used as a loading control. (C,D) Bar graphs representing the densitometric analysis of three independent experiments from Western blot (B) using the Lab image software. Differences between two groups were analyzed by the two-tailed Student's t -test and of more than two groups by One-way ANOVA with Bonferroni posttest ( n = 3) and the values were expressed as means ± SEM. Asterisk indicates the significance ( *** P

    Journal: Frontiers in Physiology

    Article Title: Immunosuppressant MPA Modulates Tight Junction through Epigenetic Activation of MLCK/MLC-2 Pathway via p38MAPK

    doi: 10.3389/fphys.2015.00381

    Figure Lengend Snippet: MPA treatment increases global histone acetylation in Caco-2 cells . (A) Dot-blot analysis; Caco-2 cells were cultured in six-well plates and post confluence monolayers were treated either with 10 μM MPA or DMSO for indicated time points. Total proteins were extracted and a volume of 2 μl from each time point was spotted on nitrocellulose membrane, dried and then probed with anti-H3K9ac and anti-H4K8ac antibodies. (B) Western blot analysis; Total cell proteins were resolved on 12.5% SDS-PAGE gels and, immunoblotted using anti-H3K9ac and anti-H4K8ac specific antibodies. β-actin was used as a loading control. (C,D) Bar graphs representing the densitometric analysis of three independent experiments from Western blot (B) using the Lab image software. Differences between two groups were analyzed by the two-tailed Student's t -test and of more than two groups by One-way ANOVA with Bonferroni posttest ( n = 3) and the values were expressed as means ± SEM. Asterisk indicates the significance ( *** P

    Article Snippet: Blocked membranes were washed twice in TBS-T for 5 min, then incubated with following antibodies: 1:10,000 dilution of mouse monoclonal anti-MLCK antibody (Sigma, Mannheim, Germany), 1:500 dilution of a mouse monoclonal anti-MLC-2 antibody (Sigma, Mannheim, Germany), 1:1000 rabbit anti-phospho MLC-2 antibody (Cell Signaling, Beverly, USA), 1 μg/mL rabbit anti-ZO-1, 0.5 μg/mL mouse anti-occludin (Zymed, CA, USA), 1:500 dilution of rabbit anti-H3K9ac (abcam), 1:250 dilution of H4K8ac (diagenode), or 1:5000 anti-β actin (Sigma, Mannheim, Germany) in 5% BSA in TBS-T for overnight at 4°C.

    Techniques: Dot Blot, Cell Culture, Western Blot, SDS Page, Software, Two Tailed Test

    With the intrathecal delivery method used, HDACI did not appear to have a measurable effect on dorsal root ganglion (DRG) acetylation, suggesting that the mechanism of action was mostly central. (A) Representative Western blot of single rat L5 DRG after nerve transection and intrathecal vehicle or HDACi treatment. Total H3 was used as a loading control for acetylated H3K9. (B) Quantification of global H3K9ac revealed no difference between vehicle and HDACI treatment groups ( n = 4, P = not significant [n.s.]).

    Journal: Pain

    Article Title: HDAC inhibitors attenuate the development of hypersensitivity in models of neuropathic pain

    doi: 10.1016/j.pain.2013.05.021

    Figure Lengend Snippet: With the intrathecal delivery method used, HDACI did not appear to have a measurable effect on dorsal root ganglion (DRG) acetylation, suggesting that the mechanism of action was mostly central. (A) Representative Western blot of single rat L5 DRG after nerve transection and intrathecal vehicle or HDACi treatment. Total H3 was used as a loading control for acetylated H3K9. (B) Quantification of global H3K9ac revealed no difference between vehicle and HDACI treatment groups ( n = 4, P = not significant [n.s.]).

    Article Snippet: Membranes were blocked in 5% milk and incubated overnight at 4 °C with a primary antibody at 1:1000 dilution (rabbit H3K9ac; Abcam, Cambridge, UK or Cell Signaling Technology, Danvers, MA) or rabbit H3 from Abcam).

    Techniques: Western Blot

    Intrathecal HDACI treatment globally increased acetylation at lysine residue 9 of histone 3 (H3K9ac) in the dorsal spinal cord. Shown are representative Western blots against H3K9ac after vehicle, MS-275 (MS30, MS60 at 30 nmol and 60 nmol/d, respectively) and MGCD0103 treatment (MG30, MG60 at 30 nmol and 60 nmol/d, respectively). Protein was obtained from ipsilateral dorsal spinal cord of animals with neuropathy as a result of spinal nerve transection (A) or antiretroviral drug injection (C). The blots were stripped and reprobed with an antibody against total H3 as a loading control. (B, D) Quantification of Western blot data using band-density analysis in ImageJ software. Significantly increased H3K9 acetylation was observed in the nerve injury model after both MS-275 and MGCD0103 treatment ( n = 4, independent-sample t tests, P

    Journal: Pain

    Article Title: HDAC inhibitors attenuate the development of hypersensitivity in models of neuropathic pain

    doi: 10.1016/j.pain.2013.05.021

    Figure Lengend Snippet: Intrathecal HDACI treatment globally increased acetylation at lysine residue 9 of histone 3 (H3K9ac) in the dorsal spinal cord. Shown are representative Western blots against H3K9ac after vehicle, MS-275 (MS30, MS60 at 30 nmol and 60 nmol/d, respectively) and MGCD0103 treatment (MG30, MG60 at 30 nmol and 60 nmol/d, respectively). Protein was obtained from ipsilateral dorsal spinal cord of animals with neuropathy as a result of spinal nerve transection (A) or antiretroviral drug injection (C). The blots were stripped and reprobed with an antibody against total H3 as a loading control. (B, D) Quantification of Western blot data using band-density analysis in ImageJ software. Significantly increased H3K9 acetylation was observed in the nerve injury model after both MS-275 and MGCD0103 treatment ( n = 4, independent-sample t tests, P

    Article Snippet: Membranes were blocked in 5% milk and incubated overnight at 4 °C with a primary antibody at 1:1000 dilution (rabbit H3K9ac; Abcam, Cambridge, UK or Cell Signaling Technology, Danvers, MA) or rabbit H3 from Abcam).

    Techniques: Western Blot, Mass Spectrometry, Injection, Software

    ChIP–quantitative polymerase chain reaction (qPCR) revealed changes in H3K9ac with drug treatment at several promoters in the spinal cord, but not the dorsal root ganglia (DRG). (A) ChIP-qPCR of dorsal ipsilateral spinal cord tissue examining H3K9ac at relevant promoters. Shown here is the ratio of enrichment of H3K9ac over H3 for a gene desert control region and the transcriptional start sites of 6 genes: Hdacs1, 2, and 11; the calcium channel subunit α2δ1 (Cacna2d1), and the transcription factors Rest and Mecp2. Histone deacetylase inhibitor resulted in increased enrichment at 4 of 6 of the genes tested, statistically significant in the case of Cacna2d1 and Mecp2 ( n = 4, independent-sample t tests, uncorrected P = .016, P = .047, respectively). (B) In the DRG, MS-275- and vehicle-treated ipsilateral DRG revealed no significant differences between treatment groups at any of the genes tested. ∗ P

    Journal: Pain

    Article Title: HDAC inhibitors attenuate the development of hypersensitivity in models of neuropathic pain

    doi: 10.1016/j.pain.2013.05.021

    Figure Lengend Snippet: ChIP–quantitative polymerase chain reaction (qPCR) revealed changes in H3K9ac with drug treatment at several promoters in the spinal cord, but not the dorsal root ganglia (DRG). (A) ChIP-qPCR of dorsal ipsilateral spinal cord tissue examining H3K9ac at relevant promoters. Shown here is the ratio of enrichment of H3K9ac over H3 for a gene desert control region and the transcriptional start sites of 6 genes: Hdacs1, 2, and 11; the calcium channel subunit α2δ1 (Cacna2d1), and the transcription factors Rest and Mecp2. Histone deacetylase inhibitor resulted in increased enrichment at 4 of 6 of the genes tested, statistically significant in the case of Cacna2d1 and Mecp2 ( n = 4, independent-sample t tests, uncorrected P = .016, P = .047, respectively). (B) In the DRG, MS-275- and vehicle-treated ipsilateral DRG revealed no significant differences between treatment groups at any of the genes tested. ∗ P

    Article Snippet: Membranes were blocked in 5% milk and incubated overnight at 4 °C with a primary antibody at 1:1000 dilution (rabbit H3K9ac; Abcam, Cambridge, UK or Cell Signaling Technology, Danvers, MA) or rabbit H3 from Abcam).

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Histone Deacetylase Assay, Mass Spectrometry

    Histone modification at the RARβ2-promoter region induced by HDAC inhibitor and ATRA treatment. (A) SAHA (10 µmol/L) treatment for 48 h, either alone or in combination with ATRA (1 µmol/L), strongly induced the hyperacetylation of histone H3 and restored RARβ2 expression in HeLa, SiHa, CaSki, and C33A cells. (B) Schematic of the RARβ2-promoter region. The exons of RARβ2 are represented by black boxes. The arrow indicates the transcription-initiation site. The PCR region (−168 to +22) for the ChIP assay included the core region of the RARE, the TATA box, and 22 bp of exon 1. (C) Representative ChIP-PCR data. PCR products were visualized via a 1% agarose gel stained with ethidium bromide. (D) DNA samples from the anti-H3K9ac IP as well as the input material and the mock immunoprecipitation samples were quantified by real-time PCR. Treatment with either 10 µmol/L SAHA or 3 mmol/L VPA led to a significant increase in RARβ2-RARE enrichment. The greatest increase in RARβ2-RARE enrichment was observed with the combination treatment. * P

    Journal: PLoS ONE

    Article Title: Targeting of Histone Deacetylases to Reactivate Tumour Suppressor Genes and Its Therapeutic Potential in a Human Cervical Cancer Xenograft Model

    doi: 10.1371/journal.pone.0080657

    Figure Lengend Snippet: Histone modification at the RARβ2-promoter region induced by HDAC inhibitor and ATRA treatment. (A) SAHA (10 µmol/L) treatment for 48 h, either alone or in combination with ATRA (1 µmol/L), strongly induced the hyperacetylation of histone H3 and restored RARβ2 expression in HeLa, SiHa, CaSki, and C33A cells. (B) Schematic of the RARβ2-promoter region. The exons of RARβ2 are represented by black boxes. The arrow indicates the transcription-initiation site. The PCR region (−168 to +22) for the ChIP assay included the core region of the RARE, the TATA box, and 22 bp of exon 1. (C) Representative ChIP-PCR data. PCR products were visualized via a 1% agarose gel stained with ethidium bromide. (D) DNA samples from the anti-H3K9ac IP as well as the input material and the mock immunoprecipitation samples were quantified by real-time PCR. Treatment with either 10 µmol/L SAHA or 3 mmol/L VPA led to a significant increase in RARβ2-RARE enrichment. The greatest increase in RARβ2-RARE enrichment was observed with the combination treatment. * P

    Article Snippet: Chromatin immunoprecipitation assay Chromatin immunoprecipitation (ChIP) was performed using the EZ-ChIP Assay kit (Millipore, USA) with a ChIP-grade antibody to acetylated histone H3 (H3K9ac, Abcam).

    Techniques: Modification, Expressing, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Staining, Immunoprecipitation, Real-time Polymerase Chain Reaction

    Immunohistochemistry of AcH3, RARβ2, involucrin, and loricrin in tumour xenografts. Treatment with VPA alone significantly increased the level of histone H3 acetylation, restored RARβ2 expression, and induced the expression of the terminal differentiation markers involucrin and loricrin, while treatment with the combination of VPA and ATRA led to greater effect in reactivating these genes expression compared to either single drug treatment. Little positive cells were observed in tissues from the ATRA-treated group.

    Journal: PLoS ONE

    Article Title: Targeting of Histone Deacetylases to Reactivate Tumour Suppressor Genes and Its Therapeutic Potential in a Human Cervical Cancer Xenograft Model

    doi: 10.1371/journal.pone.0080657

    Figure Lengend Snippet: Immunohistochemistry of AcH3, RARβ2, involucrin, and loricrin in tumour xenografts. Treatment with VPA alone significantly increased the level of histone H3 acetylation, restored RARβ2 expression, and induced the expression of the terminal differentiation markers involucrin and loricrin, while treatment with the combination of VPA and ATRA led to greater effect in reactivating these genes expression compared to either single drug treatment. Little positive cells were observed in tissues from the ATRA-treated group.

    Article Snippet: Chromatin immunoprecipitation assay Chromatin immunoprecipitation (ChIP) was performed using the EZ-ChIP Assay kit (Millipore, USA) with a ChIP-grade antibody to acetylated histone H3 (H3K9ac, Abcam).

    Techniques: Immunohistochemistry, Expressing

    VPA and ATRA promote expression of TSGs. (A) A ChIP assay was used to examine the effect of VPA and ATRA on the level of histone acetylation in the RARβ2-RARE region. (B) A significant increase in RARβ2-RARE enrichment was observed in tumors treated with a combination of VPA and ATRA, indicating that combination treatment restores RARβ2 expression via epigenetic modification. (C) VPA and ATRA restored expression of RARβ2, sequentially enhancing the expression of E-cadherin, involucrin, and loricrin, based on Q-PCR. (D) Immunoblot analysis further showed reactivation of TSGs after treatment with a combination of VPA and ATRA. This treatment resulted in the terminal differentiation and partial apoptosis of the tumour cells in the xenografts. * P

    Journal: PLoS ONE

    Article Title: Targeting of Histone Deacetylases to Reactivate Tumour Suppressor Genes and Its Therapeutic Potential in a Human Cervical Cancer Xenograft Model

    doi: 10.1371/journal.pone.0080657

    Figure Lengend Snippet: VPA and ATRA promote expression of TSGs. (A) A ChIP assay was used to examine the effect of VPA and ATRA on the level of histone acetylation in the RARβ2-RARE region. (B) A significant increase in RARβ2-RARE enrichment was observed in tumors treated with a combination of VPA and ATRA, indicating that combination treatment restores RARβ2 expression via epigenetic modification. (C) VPA and ATRA restored expression of RARβ2, sequentially enhancing the expression of E-cadherin, involucrin, and loricrin, based on Q-PCR. (D) Immunoblot analysis further showed reactivation of TSGs after treatment with a combination of VPA and ATRA. This treatment resulted in the terminal differentiation and partial apoptosis of the tumour cells in the xenografts. * P

    Article Snippet: Chromatin immunoprecipitation assay Chromatin immunoprecipitation (ChIP) was performed using the EZ-ChIP Assay kit (Millipore, USA) with a ChIP-grade antibody to acetylated histone H3 (H3K9ac, Abcam).

    Techniques: Expressing, Chromatin Immunoprecipitation, Modification, Polymerase Chain Reaction