anti-histone h3 Search Results


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  • 99
    Millipore histone h4
    Apoptotic cells exhibit reduced staining for acetylated histone H4. Immunofluorescent double labeling of cells in the ganglion cell layer of a DBA/2J mouse. Frozen sections were double labeled with antibodies against the histone variant γH2AX
    Histone H4, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 776 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Abcam anti histone h4
    Effects of neonatal exposure to sevoflurane and sodium butyrate (NaB) treatment on expression of hippocampal acetylated <t>histones</t> H3 and H4. (A, top) Representative Western blot images of acetyl-H3K9, acetyl-H3K14, and total H3 blots in the hippocampal
    Anti Histone H4, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 336 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore anti histone h3
    β-Catenin bridges AXIN2 to TCF4. (A) Western blot analysis of proteins prepared from whole cell [W], cytoplasmic [C], and nuclear [N] compartments of HCT116 cells expressing NLS-AXIN2. (B) Top , Co-immunoprecipitation/western blot analysis of proteins precipitated with anti-AXIN2 antibodies in nuclear lysates prepared from HCT116 cells. Bottom , Western blot analysis of cytoplasmic [C] and nuclear [N] fractions using α-tubulin and <t>histone</t> H3 antibodies. (C) Co-immunoprecipitation/western blot analysis of TCF4 interacting proteins. HEK293 cells were transfected with plasmids expressing the indicated cDNAs and TCF4 was precipitated with anti-TCF4 antibodies.
    Anti Histone H3, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 850 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    BioLegend purified anti histone h3
    SCFAs reduce the autoantibody response and autoimmunity in lupus-prone mice. Female MRL/ Fas lpr/lpr mice fed fiber diet were given SCFAs water (SCFAs) or plain water (Nil) starting at the age of 5 weeks and killed at 17 weeks of age. a Titers of circulating anti-dsDNA IgM, and anti-dsDNA, anti-RNP/Sm, <t>anti-histone,</t> and anti-RNA IgG1 and IgG2a (relative units, RU), as analyzed by specific ELISAs. Each symbol represents an individual mouse ( n = 4 per group, pooled from two experiments). The bars represent the mean ± SD. b ANA visualized by indirect immunofluorescence on HEp-2 cells that were incubated with sera (1:400 dilution) from the MRL/ Fas lp/lpr mice using FITC-labeled rat mAbs to mouse IgG1 and IgG2a. Data are from one of three independent experiments yielding comparable results. c , d AID and Blimp1 expression as analyzed by intracellular staining followed by fluorescence microscopy ( c ) and flow cytometry ( d ). e Spleen surface CD138 + plasmablasts/plasma cells, and intracellular CD138 + IgG1 + and CD138 + IgG2a + plasmablasts/plasma cells as analyzed by flow cytometry. Data are representative of three independent experiments yielding comparable results. f Dorsal skin lesions (left panels), kidney sections stained with H E (middle panels), and kidney sections stained with FITC-labeled rat mAbs to mouse IgG1 and IgG2a (right panels). Data are representative of three independent experiments. * p
    Purified Anti Histone H3, supplied by BioLegend, used in various techniques. Bioz Stars score: 94/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti histone h3 pser10
    ATM-mediated Mad1 and Bub1 phosphorylation functions independently to regulate the spindle checkpoint. ( A ) HeLa cells were transfected with vector, Flag-tagged WT or S314A mutants of Bub1 and treated with nocodazole for 16h. Cells were then harvested and immunoblotted with indicated antibodies. ( B ) Cells were transfected with vector, HA-tagged WT or S214A mutants of Mad1 followed by treatment with nocodazole for 16h. Cells were then harvested for immunoblotting with indicated antibodies. ( C ) Cells were transfected with vector, S214A Mad1 only, S314A Bub1 only or cotransfected with S214AMad1 and S314ABub1 plasmids. After nocodazole treatment (16h), mitotic cells were analyzed by flow cytometry-based <t>histone-H3-Ser10p</t> staining. Mean mitotic percentages of at least triplicate samples are shown, and error bars represent the variations around the averages. Statistic analyses were done by t -test and P values are presented.
    Anti Histone H3 Pser10, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Abcam antihistone h3
    HMGB1 is associated with nucleosomes spontaneously released during secondary necrosis. For induction of secondary necrosis (SN), Jurkat cells were treated with 2 μM staurosporine for 48 h. Primary necrosis was induced by heating cells at 56°C for 30 min followed by incubation for an additional hour at 37°C. Supernatants from apoptotic (top) and necrotic (bottom) Jurkat cells were collected and subjected to immune precipitations with anti-HMGB1, anti-dsDNA, <t>antihistone</t> H3, antihistone H2B, and antihistones H2A/H4 as well as with appropriate isotype control antibodies. Immune-precipitated material was then fractionated by SDS-PAGE on 12% polyacrylamide gel. Western blot analysis was performed using polyclonal anti-HMGB1 antibodies. WB, Western blotting; IP, immunoprecipitation. These experiments have been performed four times with virtually identical results.
    Antihistone H3, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology anti histone h1
    Induction of apoptosis by LA and analysis on poly(ADP-ribose) polymerase (PARP) cleavage, AIF/cytochrome c expression, and bax/bcl-2 ratio and subcellular distribution of AIF/cytochrome c by LA. (A) HL-60 cells were treated with 0, 2.5 and 5 mM LA for 24 to 48 h; LA induced cell death, evident by the flow cytometric measured sub-G1 fraction was calculated and shown as % of total cell population. (B) Western blot analysis revealed down regulation of PARP expression at accompanied by appearance of 89 kDa cleaved PARP fragment in ≥ 2.5 mM, 48 h LA treated cells. (C) AIF and cytochrome c (Cyt C) expression in 48 h LA treated cells. (D) The actin-adjusted level of bax and bcl-2 and changes in the ratio of bax to bcl-2 in HL-60 cells treated for 48 h with increasing dose of LA. (E) Subcellular distribution of immunoreactive AIF and Cyt C in the cytosol, mitochondria and nucleus in control and 24 and 48 h LA-treated HL-60 cells. Actin and <t>histone</t> was used as loading control for cytosol and nucleus fractions, respectively. For mitochondria fraction verification was performed as detailed in Methods.
    Anti Histone H1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 337 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Active Motif anti histone h1
    Association of cellular <t>histones</t> with amplified viral genomes. (A) EA.hy926-RTA cells containing latent KSHV were treated with doxycycline for 48 h or left untreated prior to IF analysis of the distribution of histones H1 and H3 in relation to KSHV RCs marked by the viral PF8 protein. Scale bars represent 5 μm. (B and C) TREx BCBL-1-RTA cells were treated with doxycycline for 0, 24, and 48 h and chromatin preparations were prepared by sonication. (B) Following DNA cleanup, the viral genome load was determined using qPCR relative to the 0-h control. (C) Chromatin preparations were subjected to ChIP for <t>histone</t> H3 or control IgG. Viral DNA was detected in immunoprecipitated material by qPCR using primers specific to the ORF47 or OriLytL region of the KSHV genome. Following subtraction of background readings, values were normalized to cellular DNA detected using primers specific to β2-microglobulin (Bm2). All data bars represent the mean from three independent experiments, while error bars signify the SEM.
    Anti Histone H1, supplied by Active Motif, used in various techniques. Bioz Stars score: 91/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Proteintech histone h3
    H2 (A)and <t>H2B</t> ubiquitination were increased in testes from RAD6B−/− males but decreased in testes from RNF8−/− males. (A) Basic nuclear proteins were isolated from total testis from WT, RNF8−/− and RAD6B−/− mice, separated using 15% SDS-PAGE, and stained with Coomassie blue. Proteins were excised from the gel and identified by MS. (B) H1 T was decreased in testes from RNF8−/− and RAD6B−/− mice and H2 A and H2B were increased in testes from RAD6B−/− males. In contrast, no significant changes were observed in RNF8−/− males. Western blots of proteins using testes from WT, RNF8−/− and RAD6B−/− mice are shown. Antibodies used are indicated, and H4 was used as a loading control. (C) The relative expression levels of H1 T, H2 A and H2B in panel B are summarized in the histogram (mean ± SEM). (D) ub-H2 A and ub-H2B were decreased in testes from RNF8−/− mice but increased in testes from RAD6B−/− mice. Western blots of proteins from testes of WT, RNF8−/− and RAD6B−/− mice are shown. Antibodies used are indicated, and H4 was used as a loading control. (E) H2 A-ub2 had almost completely disappeared from the testes of RAD6B−/− mice and was decreased significantly in the testes of RNF8−/− mice. Western blots of proteins using testes from WT, RNF8−/− and RAD6B−/− mice are shown.
    Histone H3, supplied by Proteintech, used in various techniques. Bioz Stars score: 90/100, based on 132 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck KGaA histone h3
    Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated <t>histone</t> H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p
    Histone H3, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc histone h2b
    Depletion of CRL7 SMU1 complex proteins induce mitotic defects. (A) HeLa cells were transfected with either control or SMU1 siRNAs. Cells were stained with antibody against α-tubulin to check the spindle defects (multipolar spindles) and nuclei were counterstained with DAPI to check for chromosomal defects (lagging chromosomes and anaphase bridges). Scale bar: 10 μm. (B) Quantification of results shown in A ( n =50 cells each). (C) HeLa cells were transduced with control shRNA, CUL7 shRNA, DDB1 shRNA or RNF40 shRNA. Cells were stained with antibody against α-tubulin to check the spindle defects (multipolar spindles) and nuclei were counterstained with DAPI to check for chromosomal defects (lagging chromosomes and anaphase bridges). Scale bar: 5 μm. (D) Quantification of results shown in C ( n =40 cells each). (E) Cells were transfected with <t>H2B</t> wild type (WT) and H2B K120R mutant. (F) Various mitotic abnormalities in cells expressing H2B WT and H2B K120R mutant were checked using immunofluorescence after staining with antibody against α-tubulin and DAPI. Scale bar: 5 μm. (G) Quantification of results shown in F ( n =50 cells each). Error bars indicate the mean+s.d.; *** P
    Histone H2b, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Upstate Biotechnology Inc histone h3
    Quantitative ChIP analyses of barrier-associated protein occupancy in the ankyrin-1 5′HS region. Quantitative ChIP studies were performed to examine barrier-associated protein occupancy in the ankyrin 5′HS region using K562 cell (left) and primary erythroid cell (right) chromatin. ( A ) was included as a negative control. ( B ) Methyltransferases. ChIP analyses of the ankyrin 5′HS region using antibodies against PRMT1 and PRMT4. A region of the CITED2 ) was included as a positive control, and a region of the hsSat2 was included as a negative control. ( C ) <t>Histone</t> acetylases and chromatin remodeling proteins. ChIP analyses of the ankyrin 5′HS region using antibodies against CBP, PCAF, and SMARCA4/BRG1. A region of the IL4 ), and a region of the hsSat2 was included as a negative control (–C) for all 3 antibodies. ( D ) The region and primers utilized in ChIP.
    Histone H3, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1067 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GenScript anti histone h3
    Accumulation in N. benthamiana Plants. (A) Downregulating expression of TFIIIA as well as 5S rRNA , shown by RNA gel blotting. Immunoblots of leaf extracts showed reduced levels of TFIIIA-7ZF as well as increased level of TFIIIA-9ZF upon antisense suppression of TFIIIA . <t>Histone</t> H3 served as a loading control for protein gel blots, while ethidium bromide staining of rRNAs served as a loading control for RNA gel blots. (B) upon ectopic expression of TFIIIA-7ZF but not of TFIIIA-9ZF . .
    Anti Histone H3, supplied by GenScript, used in various techniques. Bioz Stars score: 93/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Agrisera anti histone h3
    Accumulation in N. benthamiana Plants. (A) Downregulating expression of TFIIIA as well as 5S rRNA , shown by RNA gel blotting. Immunoblots of leaf extracts showed reduced levels of TFIIIA-7ZF as well as increased level of TFIIIA-9ZF upon antisense suppression of TFIIIA . <t>Histone</t> H3 served as a loading control for protein gel blots, while ethidium bromide staining of rRNAs served as a loading control for RNA gel blots. (B) upon ectopic expression of TFIIIA-7ZF but not of TFIIIA-9ZF . .
    Anti Histone H3, supplied by Agrisera, used in various techniques. Bioz Stars score: 93/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Apoptotic cells exhibit reduced staining for acetylated histone H4. Immunofluorescent double labeling of cells in the ganglion cell layer of a DBA/2J mouse. Frozen sections were double labeled with antibodies against the histone variant γH2AX

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Silencing of Fem1cR3 Gene Expression in the DBA/2J Mouse Precedes Retinal Ganglion Cell Death and Is Associated with Histone Deacetylase Activity

    doi: 10.1167/iovs.11-8872

    Figure Lengend Snippet: Apoptotic cells exhibit reduced staining for acetylated histone H4. Immunofluorescent double labeling of cells in the ganglion cell layer of a DBA/2J mouse. Frozen sections were double labeled with antibodies against the histone variant γH2AX

    Article Snippet: Similarly, colocalization of acetylated histone H4 (AcH4) and γH2AX showed that normal-appearing (stage I) cells had robust AcH4 labeling ( ).

    Techniques: Staining, Labeling, Variant Assay

    HDAC3 concentrates in nuclei of apoptotic cells. Immunofluorescent double labeling of cells in the ganglion cell layer of a DBA/2J mouse. Frozen sections were double labeled with antibodies against the histone variant γH2AX to identify dying cells,

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Silencing of Fem1cR3 Gene Expression in the DBA/2J Mouse Precedes Retinal Ganglion Cell Death and Is Associated with Histone Deacetylase Activity

    doi: 10.1167/iovs.11-8872

    Figure Lengend Snippet: HDAC3 concentrates in nuclei of apoptotic cells. Immunofluorescent double labeling of cells in the ganglion cell layer of a DBA/2J mouse. Frozen sections were double labeled with antibodies against the histone variant γH2AX to identify dying cells,

    Article Snippet: Similarly, colocalization of acetylated histone H4 (AcH4) and γH2AX showed that normal-appearing (stage I) cells had robust AcH4 labeling ( ).

    Techniques: Labeling, Variant Assay

    Effects of neonatal exposure to sevoflurane and sodium butyrate (NaB) treatment on expression of hippocampal acetylated histones H3 and H4. (A, top) Representative Western blot images of acetyl-H3K9, acetyl-H3K14, and total H3 blots in the hippocampal

    Journal: Neurobiology of disease

    Article Title: Role of Histone Acetylation in Long-term Neurobehavioral Effects of Neonatal Exposure to Sevoflurane in Rats

    doi: 10.1016/j.nbd.2016.03.017

    Figure Lengend Snippet: Effects of neonatal exposure to sevoflurane and sodium butyrate (NaB) treatment on expression of hippocampal acetylated histones H3 and H4. (A, top) Representative Western blot images of acetyl-H3K9, acetyl-H3K14, and total H3 blots in the hippocampal

    Article Snippet: The sections were rinsed with PBS three times for 5 min to wash off the OCT, mounted with 10% normal goat serum for 1 h at room temperature, and then incubated with anti-histone H3 (acetyl K9; 1:500; Abcam), anti-histone H3 (acetyl K14; 1:500; Abcam), anti-histone H4 (acetyl K5; 1:500; Abcam), or anti-histone H4 (acetyl K12; 1:500; Abcam) overnight at 4 °C.

    Techniques: Expressing, Western Blot

    β-Catenin bridges AXIN2 to TCF4. (A) Western blot analysis of proteins prepared from whole cell [W], cytoplasmic [C], and nuclear [N] compartments of HCT116 cells expressing NLS-AXIN2. (B) Top , Co-immunoprecipitation/western blot analysis of proteins precipitated with anti-AXIN2 antibodies in nuclear lysates prepared from HCT116 cells. Bottom , Western blot analysis of cytoplasmic [C] and nuclear [N] fractions using α-tubulin and histone H3 antibodies. (C) Co-immunoprecipitation/western blot analysis of TCF4 interacting proteins. HEK293 cells were transfected with plasmids expressing the indicated cDNAs and TCF4 was precipitated with anti-TCF4 antibodies.

    Journal: Biochemical and biophysical research communications

    Article Title: Nuclear AXIN2 represses MYC gene expression

    doi: 10.1016/j.bbrc.2013.11.089

    Figure Lengend Snippet: β-Catenin bridges AXIN2 to TCF4. (A) Western blot analysis of proteins prepared from whole cell [W], cytoplasmic [C], and nuclear [N] compartments of HCT116 cells expressing NLS-AXIN2. (B) Top , Co-immunoprecipitation/western blot analysis of proteins precipitated with anti-AXIN2 antibodies in nuclear lysates prepared from HCT116 cells. Bottom , Western blot analysis of cytoplasmic [C] and nuclear [N] fractions using α-tubulin and histone H3 antibodies. (C) Co-immunoprecipitation/western blot analysis of TCF4 interacting proteins. HEK293 cells were transfected with plasmids expressing the indicated cDNAs and TCF4 was precipitated with anti-TCF4 antibodies.

    Article Snippet: Each lane of an 8% polyacrylamide gel contained 20 μg of protein lysates and the blots were probed with the following primary antibodies: anti-AXIN2 (Santa Cruz, sc-20784, 1:250), anti-α-tubulin (Sigma, T9026, 1:1000), anti-histone H3 (Millipore, 07-690, 1:25000), anti-TCF4 (Millipore, 05-511, 1:1000), and anti-β-catenin (BD Transduction, 610154, 1:1000).

    Techniques: Western Blot, Expressing, Immunoprecipitation, Transfection

    NUE automethylation regulates its activity towards histones. (A) NUE automethylation is SAM-dependent. Recombinant GST-NUE was incubated in the presence of 14 C-SAM and increasing amounts of non-radioactive SAM for 1 h at 30°C. Samples were then separated by SDS-PAGE electrophoresis and stained with Coomassie blue prior to gel dehydration and 24 h exposure to capture radioactive events. (B) NUE automethylates outside the GST moiety. GST-NUE was incubated with chlamydial protein GST-CT529 (1∶4 or 1∶2 molar ratio) or GST alone (1∶5, 1∶3 or 1∶1 molar ratio). Samples were then treated as in panel A. (C) NUE mutants have diminished activity towards histones. NUE, NUEΔ12, NUEGG (KK→GG) or NUEAA (KK→AA) were incubated in the presence of core histones for 1 h at 30°C. Samples were then treated as in panel A. All gels are representative of at least 2 independent experiments.

    Journal: PLoS Pathogens

    Article Title: Histone Methylation by NUE, a Novel Nuclear Effector of the Intracellular Pathogen Chlamydia trachomatis

    doi: 10.1371/journal.ppat.1000995

    Figure Lengend Snippet: NUE automethylation regulates its activity towards histones. (A) NUE automethylation is SAM-dependent. Recombinant GST-NUE was incubated in the presence of 14 C-SAM and increasing amounts of non-radioactive SAM for 1 h at 30°C. Samples were then separated by SDS-PAGE electrophoresis and stained with Coomassie blue prior to gel dehydration and 24 h exposure to capture radioactive events. (B) NUE automethylates outside the GST moiety. GST-NUE was incubated with chlamydial protein GST-CT529 (1∶4 or 1∶2 molar ratio) or GST alone (1∶5, 1∶3 or 1∶1 molar ratio). Samples were then treated as in panel A. (C) NUE mutants have diminished activity towards histones. NUE, NUEΔ12, NUEGG (KK→GG) or NUEAA (KK→AA) were incubated in the presence of core histones for 1 h at 30°C. Samples were then treated as in panel A. All gels are representative of at least 2 independent experiments.

    Article Snippet: Rabbit polyclonal anti-Suv39H1 antibodies were from Upstate (#07-550), anti-Histone H3 antibodies from Sigma (H0164).

    Techniques: Activity Assay, Recombinant, Incubation, SDS Page, Electrophoresis, Staining

    NUE methylates mammalian histones in vitro . (A–C) Recombinant GST-NUE was purified from E. coli and incubated with either core histones (A), individual recombinant histones (B) or recombinant His-tagged chlamydial protein Hc1 (C) in the presence of 14 C-SAM for 1 h at 30°C. Samples were then separated on a 15% SDS-PAGE gel and stained with Coomassie blue prior to gel dehydration and 24 h exposure to capture radioactive events. PRMT1, a histone H4 methyltransferase, was used as a positive control in panel A. Various concentrations of histone H2B were used as a positive control in panel C.

    Journal: PLoS Pathogens

    Article Title: Histone Methylation by NUE, a Novel Nuclear Effector of the Intracellular Pathogen Chlamydia trachomatis

    doi: 10.1371/journal.ppat.1000995

    Figure Lengend Snippet: NUE methylates mammalian histones in vitro . (A–C) Recombinant GST-NUE was purified from E. coli and incubated with either core histones (A), individual recombinant histones (B) or recombinant His-tagged chlamydial protein Hc1 (C) in the presence of 14 C-SAM for 1 h at 30°C. Samples were then separated on a 15% SDS-PAGE gel and stained with Coomassie blue prior to gel dehydration and 24 h exposure to capture radioactive events. PRMT1, a histone H4 methyltransferase, was used as a positive control in panel A. Various concentrations of histone H2B were used as a positive control in panel C.

    Article Snippet: Rabbit polyclonal anti-Suv39H1 antibodies were from Upstate (#07-550), anti-Histone H3 antibodies from Sigma (H0164).

    Techniques: In Vitro, Recombinant, Purification, Incubation, SDS Page, Staining, Positive Control

    NUE is found in the nucleus of infected cells and associates with chromatin. (A) HeLa cells were infected with C. trachomatis for 20, 25, 30 or 40 h. Nuclei were isolated as described in the Method section and both cytosolic and nuclear fractions were analyzed by Western blot using anti-NUE antibody. The membrane was stripped and re-probed for GDIβ as a cytosolic marker, PARP1 as a nuclear marker and the chlamydial protein EF-Tu to ensure the absence of bacteria in the nuclear fraction. Blots are representative of 2 separate experiments. (B) A schematic of the extraction protocol used to detect chromatin-associated proteins. (C) HeLa cells were transfected with GFP-NUE or GFP only as a negative control. Nuclei were separated from the cytosolic fraction of transfected HeLa cells. The nuclei were resuspended in 3 mM EDTA to extract soluble nuclear proteins versus chromatin-associated proteins. Samples were analyzed by Western blot using anti-GFP. HeLa cells transfected with Suv39H1 were used as a positive control and anti-Suv39H1 was used to detect the protein. Histone H3 was used to ensure chromatin enrichment in our chromatin fraction. Blots are representative of 3 separate experiments. (D) HeLa cells were infected with C. trachomatis for 40 h and analyzed for chromatin-association of NUE as in (C). Histone H3 was used to demonstrate purity of our chromatin-associated fractions and EF-Tu was used to control for bacterial contamination. Blots are representative of 2 separate experiments.

    Journal: PLoS Pathogens

    Article Title: Histone Methylation by NUE, a Novel Nuclear Effector of the Intracellular Pathogen Chlamydia trachomatis

    doi: 10.1371/journal.ppat.1000995

    Figure Lengend Snippet: NUE is found in the nucleus of infected cells and associates with chromatin. (A) HeLa cells were infected with C. trachomatis for 20, 25, 30 or 40 h. Nuclei were isolated as described in the Method section and both cytosolic and nuclear fractions were analyzed by Western blot using anti-NUE antibody. The membrane was stripped and re-probed for GDIβ as a cytosolic marker, PARP1 as a nuclear marker and the chlamydial protein EF-Tu to ensure the absence of bacteria in the nuclear fraction. Blots are representative of 2 separate experiments. (B) A schematic of the extraction protocol used to detect chromatin-associated proteins. (C) HeLa cells were transfected with GFP-NUE or GFP only as a negative control. Nuclei were separated from the cytosolic fraction of transfected HeLa cells. The nuclei were resuspended in 3 mM EDTA to extract soluble nuclear proteins versus chromatin-associated proteins. Samples were analyzed by Western blot using anti-GFP. HeLa cells transfected with Suv39H1 were used as a positive control and anti-Suv39H1 was used to detect the protein. Histone H3 was used to ensure chromatin enrichment in our chromatin fraction. Blots are representative of 3 separate experiments. (D) HeLa cells were infected with C. trachomatis for 40 h and analyzed for chromatin-association of NUE as in (C). Histone H3 was used to demonstrate purity of our chromatin-associated fractions and EF-Tu was used to control for bacterial contamination. Blots are representative of 2 separate experiments.

    Article Snippet: Rabbit polyclonal anti-Suv39H1 antibodies were from Upstate (#07-550), anti-Histone H3 antibodies from Sigma (H0164).

    Techniques: Infection, Isolation, Western Blot, Marker, Transfection, Negative Control, Positive Control

    SCFAs reduce the autoantibody response and autoimmunity in lupus-prone mice. Female MRL/ Fas lpr/lpr mice fed fiber diet were given SCFAs water (SCFAs) or plain water (Nil) starting at the age of 5 weeks and killed at 17 weeks of age. a Titers of circulating anti-dsDNA IgM, and anti-dsDNA, anti-RNP/Sm, anti-histone, and anti-RNA IgG1 and IgG2a (relative units, RU), as analyzed by specific ELISAs. Each symbol represents an individual mouse ( n = 4 per group, pooled from two experiments). The bars represent the mean ± SD. b ANA visualized by indirect immunofluorescence on HEp-2 cells that were incubated with sera (1:400 dilution) from the MRL/ Fas lp/lpr mice using FITC-labeled rat mAbs to mouse IgG1 and IgG2a. Data are from one of three independent experiments yielding comparable results. c , d AID and Blimp1 expression as analyzed by intracellular staining followed by fluorescence microscopy ( c ) and flow cytometry ( d ). e Spleen surface CD138 + plasmablasts/plasma cells, and intracellular CD138 + IgG1 + and CD138 + IgG2a + plasmablasts/plasma cells as analyzed by flow cytometry. Data are representative of three independent experiments yielding comparable results. f Dorsal skin lesions (left panels), kidney sections stained with H E (middle panels), and kidney sections stained with FITC-labeled rat mAbs to mouse IgG1 and IgG2a (right panels). Data are representative of three independent experiments. * p

    Journal: Nature Communications

    Article Title: B cell-intrinsic epigenetic modulation of antibody responses by dietary fiber-derived short-chain fatty acids

    doi: 10.1038/s41467-019-13603-6

    Figure Lengend Snippet: SCFAs reduce the autoantibody response and autoimmunity in lupus-prone mice. Female MRL/ Fas lpr/lpr mice fed fiber diet were given SCFAs water (SCFAs) or plain water (Nil) starting at the age of 5 weeks and killed at 17 weeks of age. a Titers of circulating anti-dsDNA IgM, and anti-dsDNA, anti-RNP/Sm, anti-histone, and anti-RNA IgG1 and IgG2a (relative units, RU), as analyzed by specific ELISAs. Each symbol represents an individual mouse ( n = 4 per group, pooled from two experiments). The bars represent the mean ± SD. b ANA visualized by indirect immunofluorescence on HEp-2 cells that were incubated with sera (1:400 dilution) from the MRL/ Fas lp/lpr mice using FITC-labeled rat mAbs to mouse IgG1 and IgG2a. Data are from one of three independent experiments yielding comparable results. c , d AID and Blimp1 expression as analyzed by intracellular staining followed by fluorescence microscopy ( c ) and flow cytometry ( d ). e Spleen surface CD138 + plasmablasts/plasma cells, and intracellular CD138 + IgG1 + and CD138 + IgG2a + plasmablasts/plasma cells as analyzed by flow cytometry. Data are representative of three independent experiments yielding comparable results. f Dorsal skin lesions (left panels), kidney sections stained with H E (middle panels), and kidney sections stained with FITC-labeled rat mAbs to mouse IgG1 and IgG2a (right panels). Data are representative of three independent experiments. * p

    Article Snippet: After blocking and overnight incubation at 4 °C with anti-AID (ZA001, Invitrogen), anti-Blimp1 (6D3, eBioscience), anti-acetyl-histone H3 (H3K9ac/K14ac, 17–615, Millipore), anti-histone H3 (601901, BioLegend), or anti-β-Actin mAb (AC-15, Sigma-Aldrich), the membranes were incubated with HRP-conjugated secondary Abs.

    Techniques: Mouse Assay, Immunofluorescence, Incubation, Labeling, Expressing, Staining, Fluorescence, Microscopy, Flow Cytometry, Cytometry

    SCFAs increase expression of Aicda- and Prdm1- targeting miRNAs by enhancing histone acetylation of related miRNA host genes. a Primary mouse B cells, CH12F3 B cells, and human B cells were stimulated with indicated stimuli for 60 h in the presence of nil, butyrate (500 μM), or propionate (2000 μM). Ex vivo B cells were isolated from mice injected with NP-LPS and on plain water or SCFA water for 21 days. Acetylated-histone H3 (H3K9ac), histone H3, and β-actin proteins as detected by immunoblotting. Data are one representative of three independent experiments. b Relative densities of the Ac-histone H3 bands normalized to histone H3 and β - actin levels. c , d B cells stimulated with LPS plus IL-4 in the presence of nil or butyrate for 60 h. Expression of the Aicda- and Prdm1- targeting miRNAs, and irrelevant miRNAs as analyzed by qRT-PCR ( c ). Relative abundance of H3K9ac/K14ac in miRNA host genes (HGs) as analyzed by ChIP-qPCR ( d ). e Schematic diagram of the luciferase reporter constructs-containing 3′UTRs of Aicda and Prdm1 mRNAs and their mutant (mut) counterparts. f Surface CD19 and IgA on CH12F3 B cells stimulated for 96 h in the presence of nil, butyrate (500 μM), propionate (2000 μM), or VPA (500 μM) as analyzed by flow cytometry. Data are one representative of 3 independent experiments yielding comparable results. g , miRNA expression in CH12F3 cells stimulated for 24 h in the presence of nil or butyrate, as analyzed by qRT-PCR. Data in c , d , and g are ratios to stimulated B cells cultured with nil (set as 1; means ± SEM of three independent experiments). h Luciferase activity in CH12F3 cells transfected with luciferase reporter vectors-containing wild-type or mutated Aicda or Prdm1 3′UTRs after 24 h treatment with nil or butyrate. Luciferase activity was measured 6 h after transfection, and normalized relative to luciferase activity in B cells cultured with nil. Data in h are ratios to transfected B cells cultured with nil (set as 100%; means ± SEM of three independent experiments). * p

    Journal: Nature Communications

    Article Title: B cell-intrinsic epigenetic modulation of antibody responses by dietary fiber-derived short-chain fatty acids

    doi: 10.1038/s41467-019-13603-6

    Figure Lengend Snippet: SCFAs increase expression of Aicda- and Prdm1- targeting miRNAs by enhancing histone acetylation of related miRNA host genes. a Primary mouse B cells, CH12F3 B cells, and human B cells were stimulated with indicated stimuli for 60 h in the presence of nil, butyrate (500 μM), or propionate (2000 μM). Ex vivo B cells were isolated from mice injected with NP-LPS and on plain water or SCFA water for 21 days. Acetylated-histone H3 (H3K9ac), histone H3, and β-actin proteins as detected by immunoblotting. Data are one representative of three independent experiments. b Relative densities of the Ac-histone H3 bands normalized to histone H3 and β - actin levels. c , d B cells stimulated with LPS plus IL-4 in the presence of nil or butyrate for 60 h. Expression of the Aicda- and Prdm1- targeting miRNAs, and irrelevant miRNAs as analyzed by qRT-PCR ( c ). Relative abundance of H3K9ac/K14ac in miRNA host genes (HGs) as analyzed by ChIP-qPCR ( d ). e Schematic diagram of the luciferase reporter constructs-containing 3′UTRs of Aicda and Prdm1 mRNAs and their mutant (mut) counterparts. f Surface CD19 and IgA on CH12F3 B cells stimulated for 96 h in the presence of nil, butyrate (500 μM), propionate (2000 μM), or VPA (500 μM) as analyzed by flow cytometry. Data are one representative of 3 independent experiments yielding comparable results. g , miRNA expression in CH12F3 cells stimulated for 24 h in the presence of nil or butyrate, as analyzed by qRT-PCR. Data in c , d , and g are ratios to stimulated B cells cultured with nil (set as 1; means ± SEM of three independent experiments). h Luciferase activity in CH12F3 cells transfected with luciferase reporter vectors-containing wild-type or mutated Aicda or Prdm1 3′UTRs after 24 h treatment with nil or butyrate. Luciferase activity was measured 6 h after transfection, and normalized relative to luciferase activity in B cells cultured with nil. Data in h are ratios to transfected B cells cultured with nil (set as 100%; means ± SEM of three independent experiments). * p

    Article Snippet: After blocking and overnight incubation at 4 °C with anti-AID (ZA001, Invitrogen), anti-Blimp1 (6D3, eBioscience), anti-acetyl-histone H3 (H3K9ac/K14ac, 17–615, Millipore), anti-histone H3 (601901, BioLegend), or anti-β-Actin mAb (AC-15, Sigma-Aldrich), the membranes were incubated with HRP-conjugated secondary Abs.

    Techniques: Expressing, Ex Vivo, Isolation, Mouse Assay, Injection, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Luciferase, Construct, Mutagenesis, Flow Cytometry, Cytometry, Cell Culture, Activity Assay, Transfection

    ATM-mediated Mad1 and Bub1 phosphorylation functions independently to regulate the spindle checkpoint. ( A ) HeLa cells were transfected with vector, Flag-tagged WT or S314A mutants of Bub1 and treated with nocodazole for 16h. Cells were then harvested and immunoblotted with indicated antibodies. ( B ) Cells were transfected with vector, HA-tagged WT or S214A mutants of Mad1 followed by treatment with nocodazole for 16h. Cells were then harvested for immunoblotting with indicated antibodies. ( C ) Cells were transfected with vector, S214A Mad1 only, S314A Bub1 only or cotransfected with S214AMad1 and S314ABub1 plasmids. After nocodazole treatment (16h), mitotic cells were analyzed by flow cytometry-based histone-H3-Ser10p staining. Mean mitotic percentages of at least triplicate samples are shown, and error bars represent the variations around the averages. Statistic analyses were done by t -test and P values are presented.

    Journal: Carcinogenesis

    Article Title: ATM-mediated Mad1 Serine 214 phosphorylation regulates Mad1 dimerization and the spindle assembly checkpoint

    doi: 10.1093/carcin/bgu087

    Figure Lengend Snippet: ATM-mediated Mad1 and Bub1 phosphorylation functions independently to regulate the spindle checkpoint. ( A ) HeLa cells were transfected with vector, Flag-tagged WT or S314A mutants of Bub1 and treated with nocodazole for 16h. Cells were then harvested and immunoblotted with indicated antibodies. ( B ) Cells were transfected with vector, HA-tagged WT or S214A mutants of Mad1 followed by treatment with nocodazole for 16h. Cells were then harvested for immunoblotting with indicated antibodies. ( C ) Cells were transfected with vector, S214A Mad1 only, S314A Bub1 only or cotransfected with S214AMad1 and S314ABub1 plasmids. After nocodazole treatment (16h), mitotic cells were analyzed by flow cytometry-based histone-H3-Ser10p staining. Mean mitotic percentages of at least triplicate samples are shown, and error bars represent the variations around the averages. Statistic analyses were done by t -test and P values are presented.

    Article Snippet: The anti-Flag antibody was obtained from OriGENE (Rockville, MD), and anti-histone-H3-Ser10p was from Millipore (Billerica, MA).

    Techniques: Transfection, Plasmid Preparation, Flow Cytometry, Cytometry, Staining

    HMGB1 is associated with nucleosomes spontaneously released during secondary necrosis. For induction of secondary necrosis (SN), Jurkat cells were treated with 2 μM staurosporine for 48 h. Primary necrosis was induced by heating cells at 56°C for 30 min followed by incubation for an additional hour at 37°C. Supernatants from apoptotic (top) and necrotic (bottom) Jurkat cells were collected and subjected to immune precipitations with anti-HMGB1, anti-dsDNA, antihistone H3, antihistone H2B, and antihistones H2A/H4 as well as with appropriate isotype control antibodies. Immune-precipitated material was then fractionated by SDS-PAGE on 12% polyacrylamide gel. Western blot analysis was performed using polyclonal anti-HMGB1 antibodies. WB, Western blotting; IP, immunoprecipitation. These experiments have been performed four times with virtually identical results.

    Journal: The Journal of Experimental Medicine

    Article Title: Induction of inflammatory and immune responses by HMGB1-nucleosome complexes: implications for the pathogenesis of SLE

    doi: 10.1084/jem.20081165

    Figure Lengend Snippet: HMGB1 is associated with nucleosomes spontaneously released during secondary necrosis. For induction of secondary necrosis (SN), Jurkat cells were treated with 2 μM staurosporine for 48 h. Primary necrosis was induced by heating cells at 56°C for 30 min followed by incubation for an additional hour at 37°C. Supernatants from apoptotic (top) and necrotic (bottom) Jurkat cells were collected and subjected to immune precipitations with anti-HMGB1, anti-dsDNA, antihistone H3, antihistone H2B, and antihistones H2A/H4 as well as with appropriate isotype control antibodies. Immune-precipitated material was then fractionated by SDS-PAGE on 12% polyacrylamide gel. Western blot analysis was performed using polyclonal anti-HMGB1 antibodies. WB, Western blotting; IP, immunoprecipitation. These experiments have been performed four times with virtually identical results.

    Article Snippet: In brief, 7.5 μg of anti-HMGB1 (R & D Systems), anti-dsDNA (33H11; provided by T. Winkler, University of Erlangen-Nürnberg, Erlangen, Germany), antihistone pan (Roche), antihistone KM1 and KM2 (provided by J. Berden, Nijmegen Medical Center, Nijmegen, Netherlands), and antihistone H3 (Abcam) antibodies and 7.5 μg/ml of appropriate isotype control antibodies were covalently coupled to protein G–Sepharose beads and incubated either with serum samples or cell culture supernatants of apoptotic or necrotic Jurkat cells at 4°C for 4 h. Precipitated proteins were analyzed by Western blotting with polyclonal anti-HMGB1 antibodies (Millipore).

    Techniques: Incubation, SDS Page, Western Blot, Immunoprecipitation

    Induction of apoptosis by LA and analysis on poly(ADP-ribose) polymerase (PARP) cleavage, AIF/cytochrome c expression, and bax/bcl-2 ratio and subcellular distribution of AIF/cytochrome c by LA. (A) HL-60 cells were treated with 0, 2.5 and 5 mM LA for 24 to 48 h; LA induced cell death, evident by the flow cytometric measured sub-G1 fraction was calculated and shown as % of total cell population. (B) Western blot analysis revealed down regulation of PARP expression at accompanied by appearance of 89 kDa cleaved PARP fragment in ≥ 2.5 mM, 48 h LA treated cells. (C) AIF and cytochrome c (Cyt C) expression in 48 h LA treated cells. (D) The actin-adjusted level of bax and bcl-2 and changes in the ratio of bax to bcl-2 in HL-60 cells treated for 48 h with increasing dose of LA. (E) Subcellular distribution of immunoreactive AIF and Cyt C in the cytosol, mitochondria and nucleus in control and 24 and 48 h LA-treated HL-60 cells. Actin and histone was used as loading control for cytosol and nucleus fractions, respectively. For mitochondria fraction verification was performed as detailed in Methods.

    Journal: Journal of Hematology and Oncology

    Article Title: Regulation of cell cycle transition and induction of apoptosis in HL-60 leukemia cells by lipoic acid: role in cancer prevention and therapy

    doi: 10.1186/1756-8722-1-4

    Figure Lengend Snippet: Induction of apoptosis by LA and analysis on poly(ADP-ribose) polymerase (PARP) cleavage, AIF/cytochrome c expression, and bax/bcl-2 ratio and subcellular distribution of AIF/cytochrome c by LA. (A) HL-60 cells were treated with 0, 2.5 and 5 mM LA for 24 to 48 h; LA induced cell death, evident by the flow cytometric measured sub-G1 fraction was calculated and shown as % of total cell population. (B) Western blot analysis revealed down regulation of PARP expression at accompanied by appearance of 89 kDa cleaved PARP fragment in ≥ 2.5 mM, 48 h LA treated cells. (C) AIF and cytochrome c (Cyt C) expression in 48 h LA treated cells. (D) The actin-adjusted level of bax and bcl-2 and changes in the ratio of bax to bcl-2 in HL-60 cells treated for 48 h with increasing dose of LA. (E) Subcellular distribution of immunoreactive AIF and Cyt C in the cytosol, mitochondria and nucleus in control and 24 and 48 h LA-treated HL-60 cells. Actin and histone was used as loading control for cytosol and nucleus fractions, respectively. For mitochondria fraction verification was performed as detailed in Methods.

    Article Snippet: Primary antibodies like anti-Rb, anti-E2F, anti-cyclin B1, anti-cyclin D, anti-cyclin E, anti-cdk1, anti-cdk2, anti-AIF, anti-cytochrome c, anti-bcl-2, anti-bax, anti-actin, anti-histone H1, and secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques: Expressing, Flow Cytometry, Western Blot

    Decreased acetylation of histone H3 at the mGlu2 promoter in prefrontal cortex of treated, but not untreated, schizophrenic subjects ( a,b ) Digested chromatin was immunoprecipitated with antibody recognizing acetyl-histone H3 (H3ac), and the level of association of the 5HT2A, 5HT2C, mGlu2, or mGlu3 promoters was measured by qPCR. The promoter of β 2 - microglobulin ( B2M ) was included as internal control. Experiments were performed in frontal cortex of untreated schizophrenic subjects and matched controls ( a ), and in frontal cortex of atypical antipsychotic-treated schizophrenic subjects and matched controls ( b ). H3ac at mGlu2 promoter in treated schizophrenics: t = 3.96. The α value was corrected for multiple independent null hypotheses by using the Holm’s sequentially rejective Bonferroni method. * P

    Journal: Nature neuroscience

    Article Title: HDAC2 regulates atypical antipsychotic responses through the modulation of mGlu2 promoter activity

    doi: 10.1038/nn.3181

    Figure Lengend Snippet: Decreased acetylation of histone H3 at the mGlu2 promoter in prefrontal cortex of treated, but not untreated, schizophrenic subjects ( a,b ) Digested chromatin was immunoprecipitated with antibody recognizing acetyl-histone H3 (H3ac), and the level of association of the 5HT2A, 5HT2C, mGlu2, or mGlu3 promoters was measured by qPCR. The promoter of β 2 - microglobulin ( B2M ) was included as internal control. Experiments were performed in frontal cortex of untreated schizophrenic subjects and matched controls ( a ), and in frontal cortex of atypical antipsychotic-treated schizophrenic subjects and matched controls ( b ). H3ac at mGlu2 promoter in treated schizophrenics: t = 3.96. The α value was corrected for multiple independent null hypotheses by using the Holm’s sequentially rejective Bonferroni method. * P

    Article Snippet: The following primary antibodies were used: HDAC1 (mouse brain: Cell Signaling 5356, 1:1000; postmortem human brain: Santa Cruz sc-6299, 1:200), HDAC2 (mouse brain: Abcam 12169, 1:400; postmortem human brain: Abcam 32117, 1:2000), HDAC4 (mouse brain: Abcam 1437, 1:400; postmortem human brain: Santa Cruz sc-5245, 1:200), acetyl-histone H3 (Millipore 06-599B, 1:400), FLAG (Sigma F7425, 1:1000), β-actin (Sigma A1978, 1:200000), GAPDH (Cell Signaling 14C10, 1:1000), α-tubulin (Cell Signaling 11H10, 1:10000), and histone H1 (Santa Cruz sc-10806, 1:200).

    Techniques: Immunoprecipitation, Real-time Polymerase Chain Reaction

    Decreased acetylation of histone H3 at the mGlu2 promoter by chronic treatment with atypical antipsychotic drugs in mouse frontal cortex ( a–d ) Chronic clozapine and risperidone, but not haloperidol, modulate the expression of mGlu2 in mouse frontal cortex. Mice were chronically (21 days) injected with vehicle (black), 10 mg/kg clozapine (red), 4 mg/kg risperidone (green), or 1 mg/kg haloperidol (blue), and sacrificed one day after the last injection. ( a–c ) [ 3 H]LY341495 binding in mouse frontal cortex after vehicle or chronic clozapine ( a ), risperidone ( b ) or haloperidol ( c ). Effect of clozapine (n = 4 independent experiments performed in triplicate), F[2,95] = 65.34, P

    Journal: Nature neuroscience

    Article Title: HDAC2 regulates atypical antipsychotic responses through the modulation of mGlu2 promoter activity

    doi: 10.1038/nn.3181

    Figure Lengend Snippet: Decreased acetylation of histone H3 at the mGlu2 promoter by chronic treatment with atypical antipsychotic drugs in mouse frontal cortex ( a–d ) Chronic clozapine and risperidone, but not haloperidol, modulate the expression of mGlu2 in mouse frontal cortex. Mice were chronically (21 days) injected with vehicle (black), 10 mg/kg clozapine (red), 4 mg/kg risperidone (green), or 1 mg/kg haloperidol (blue), and sacrificed one day after the last injection. ( a–c ) [ 3 H]LY341495 binding in mouse frontal cortex after vehicle or chronic clozapine ( a ), risperidone ( b ) or haloperidol ( c ). Effect of clozapine (n = 4 independent experiments performed in triplicate), F[2,95] = 65.34, P

    Article Snippet: The following primary antibodies were used: HDAC1 (mouse brain: Cell Signaling 5356, 1:1000; postmortem human brain: Santa Cruz sc-6299, 1:200), HDAC2 (mouse brain: Abcam 12169, 1:400; postmortem human brain: Abcam 32117, 1:2000), HDAC4 (mouse brain: Abcam 1437, 1:400; postmortem human brain: Santa Cruz sc-5245, 1:200), acetyl-histone H3 (Millipore 06-599B, 1:400), FLAG (Sigma F7425, 1:1000), β-actin (Sigma A1978, 1:200000), GAPDH (Cell Signaling 14C10, 1:1000), α-tubulin (Cell Signaling 11H10, 1:10000), and histone H1 (Santa Cruz sc-10806, 1:200).

    Techniques: Expressing, Mouse Assay, Injection, Binding Assay

    Association of cellular histones with amplified viral genomes. (A) EA.hy926-RTA cells containing latent KSHV were treated with doxycycline for 48 h or left untreated prior to IF analysis of the distribution of histones H1 and H3 in relation to KSHV RCs marked by the viral PF8 protein. Scale bars represent 5 μm. (B and C) TREx BCBL-1-RTA cells were treated with doxycycline for 0, 24, and 48 h and chromatin preparations were prepared by sonication. (B) Following DNA cleanup, the viral genome load was determined using qPCR relative to the 0-h control. (C) Chromatin preparations were subjected to ChIP for histone H3 or control IgG. Viral DNA was detected in immunoprecipitated material by qPCR using primers specific to the ORF47 or OriLytL region of the KSHV genome. Following subtraction of background readings, values were normalized to cellular DNA detected using primers specific to β2-microglobulin (Bm2). All data bars represent the mean from three independent experiments, while error bars signify the SEM.

    Journal: Journal of Virology

    Article Title: Localization of Double-Strand Break Repair Proteins to Viral Replication Compartments following Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus

    doi: 10.1128/JVI.00930-17

    Figure Lengend Snippet: Association of cellular histones with amplified viral genomes. (A) EA.hy926-RTA cells containing latent KSHV were treated with doxycycline for 48 h or left untreated prior to IF analysis of the distribution of histones H1 and H3 in relation to KSHV RCs marked by the viral PF8 protein. Scale bars represent 5 μm. (B and C) TREx BCBL-1-RTA cells were treated with doxycycline for 0, 24, and 48 h and chromatin preparations were prepared by sonication. (B) Following DNA cleanup, the viral genome load was determined using qPCR relative to the 0-h control. (C) Chromatin preparations were subjected to ChIP for histone H3 or control IgG. Viral DNA was detected in immunoprecipitated material by qPCR using primers specific to the ORF47 or OriLytL region of the KSHV genome. Following subtraction of background readings, values were normalized to cellular DNA detected using primers specific to β2-microglobulin (Bm2). All data bars represent the mean from three independent experiments, while error bars signify the SEM.

    Article Snippet: The following primary antibodies were used for IF microscopy: anti-LANA (NCL-HHV8-LNA; Novacastra), anti-ORF6/SSB (provided by Gary Hayward, Johns Hopkins University, MD, USA), anti-ORF59/PF8 (in-house), anti-K8.1 (in-house), anti-MRE11 (GTX70212; Genetex), anti-NBS1 (GTX70222; Genetex), anti-RAD50 (ab89; Abcam), anti-Ku80 (GTX70276; Genetex), anti-Ku70 (ab83501; Abcam), anti-BRCA1 (sc-6954; Santa Cruz), anti-RAD51 (PC130; Merck Millipore), anti-RAD52 (sc-8350; Santa Cruz), anti-RAD54B (GTX103291; Genetex), anti-γH2AX (S139) (05-636; Merck Millipore), anti-MDC1 (in-house), anti-53BP1 (ab36823; Abcam), anti-histone H1 (39708; Active Motif), and anti-histone H3 (GTX122148; Genetex).

    Techniques: Amplification, Sonication, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Immunoprecipitation

    Tubacin enhances cell death induced by SAHA, etoposide, or doxorubicin in transformed LNCaP and MCF-7 cells. ( A ) Western blot analysis probing with antibodies against acetylated α-tubulin and acetylated histone H3 in LNCaP cells cultured for 24

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Selective inhibition of histone deacetylase 6 (HDAC6) induces DNA damage and sensitizes transformed cells to anticancer agents

    doi: 10.1073/pnas.1013754107

    Figure Lengend Snippet: Tubacin enhances cell death induced by SAHA, etoposide, or doxorubicin in transformed LNCaP and MCF-7 cells. ( A ) Western blot analysis probing with antibodies against acetylated α-tubulin and acetylated histone H3 in LNCaP cells cultured for 24

    Article Snippet: Antibodies used were HDAC6, HDAC1, HDAC3 (Santa Cruz Biotechnology), acetylated α-tubulin (Sigma), α-tubulin (Calbiochem), PARP (BD Pharmingen), γH2AX (Abcam), H2AX (Abcam), phospho-CHK2 (Cell Signaling), DDIT3 (Santa Cruz Biotechnology), GAPDH (Thermo Scientific), and Histone H3 (Active Motif).

    Techniques: Transformation Assay, Western Blot, Cell Culture

    HFS cells are resistant to cell death induced by tubacin cultured in combination with SAHA, etoposide, or doxorubicin. ( A ) Western blot analysis probing with antibodies against acetylated α-tubulin and acetylated histone H3 in HFS cells cultured

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Selective inhibition of histone deacetylase 6 (HDAC6) induces DNA damage and sensitizes transformed cells to anticancer agents

    doi: 10.1073/pnas.1013754107

    Figure Lengend Snippet: HFS cells are resistant to cell death induced by tubacin cultured in combination with SAHA, etoposide, or doxorubicin. ( A ) Western blot analysis probing with antibodies against acetylated α-tubulin and acetylated histone H3 in HFS cells cultured

    Article Snippet: Antibodies used were HDAC6, HDAC1, HDAC3 (Santa Cruz Biotechnology), acetylated α-tubulin (Sigma), α-tubulin (Calbiochem), PARP (BD Pharmingen), γH2AX (Abcam), H2AX (Abcam), phospho-CHK2 (Cell Signaling), DDIT3 (Santa Cruz Biotechnology), GAPDH (Thermo Scientific), and Histone H3 (Active Motif).

    Techniques: Cell Culture, Western Blot

    H2 (A)and H2B ubiquitination were increased in testes from RAD6B−/− males but decreased in testes from RNF8−/− males. (A) Basic nuclear proteins were isolated from total testis from WT, RNF8−/− and RAD6B−/− mice, separated using 15% SDS-PAGE, and stained with Coomassie blue. Proteins were excised from the gel and identified by MS. (B) H1 T was decreased in testes from RNF8−/− and RAD6B−/− mice and H2 A and H2B were increased in testes from RAD6B−/− males. In contrast, no significant changes were observed in RNF8−/− males. Western blots of proteins using testes from WT, RNF8−/− and RAD6B−/− mice are shown. Antibodies used are indicated, and H4 was used as a loading control. (C) The relative expression levels of H1 T, H2 A and H2B in panel B are summarized in the histogram (mean ± SEM). (D) ub-H2 A and ub-H2B were decreased in testes from RNF8−/− mice but increased in testes from RAD6B−/− mice. Western blots of proteins from testes of WT, RNF8−/− and RAD6B−/− mice are shown. Antibodies used are indicated, and H4 was used as a loading control. (E) H2 A-ub2 had almost completely disappeared from the testes of RAD6B−/− mice and was decreased significantly in the testes of RNF8−/− mice. Western blots of proteins using testes from WT, RNF8−/− and RAD6B−/− mice are shown.

    Journal: Cell Cycle

    Article Title: Function of RAD6B and RNF8 in spermatogenesis

    doi: 10.1080/15384101.2017.1361066

    Figure Lengend Snippet: H2 (A)and H2B ubiquitination were increased in testes from RAD6B−/− males but decreased in testes from RNF8−/− males. (A) Basic nuclear proteins were isolated from total testis from WT, RNF8−/− and RAD6B−/− mice, separated using 15% SDS-PAGE, and stained with Coomassie blue. Proteins were excised from the gel and identified by MS. (B) H1 T was decreased in testes from RNF8−/− and RAD6B−/− mice and H2 A and H2B were increased in testes from RAD6B−/− males. In contrast, no significant changes were observed in RNF8−/− males. Western blots of proteins using testes from WT, RNF8−/− and RAD6B−/− mice are shown. Antibodies used are indicated, and H4 was used as a loading control. (C) The relative expression levels of H1 T, H2 A and H2B in panel B are summarized in the histogram (mean ± SEM). (D) ub-H2 A and ub-H2B were decreased in testes from RNF8−/− mice but increased in testes from RAD6B−/− mice. Western blots of proteins from testes of WT, RNF8−/− and RAD6B−/− mice are shown. Antibodies used are indicated, and H4 was used as a loading control. (E) H2 A-ub2 had almost completely disappeared from the testes of RAD6B−/− mice and was decreased significantly in the testes of RNF8−/− mice. Western blots of proteins using testes from WT, RNF8−/− and RAD6B−/− mice are shown.

    Article Snippet: Proteins were separated on SDS-PAGE gels (15% SDS-PAGE, 120 V, 0–4°C) and subjected to either staining with Coomassie brilliant blue (CBB) or Western blot using the following antibodies: anti-ubiquity l -histone H2B [clone 56] (05–1312, Millipore, 1:500 dilution), anti-ubiquity l -histone H2 A [clone E6C5] (05–678, Millipore, 1:500 dilution), anti-histone H2B (15857–1-AP, Proteintech, 1:500 dilution), anti-HIST1H1 T (18188–1-AP, Proteintech, 1:500 dilution), anti-histone H4 (16047–1-AP, Proteintech, 1:500 dilution) and anti-ubiquitin (ab7780, Abcam, 1:1000 dilution) .The CBB staining gel was analyzed using liquid chromatography-mass spectrometry (LC-MS).

    Techniques: Isolation, Mouse Assay, SDS Page, Staining, Mass Spectrometry, Western Blot, Expressing

    Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p

    Journal: Biochimica et Biophysica Acta

    Article Title: Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation

    doi: 10.1016/j.bbagrm.2018.03.007

    Figure Lengend Snippet: Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p

    Article Snippet: Antibodies against acetylated histone H3 (06-599, Merck Millipore, Billerica, MA, USA), H3K27me3 (07-499, Merck Millipore), or normal rabbit IgG (12-370, Merck Millipore) were used for immunoprecipitation.

    Techniques: Binding Assay, Amplification, Chromatin Immunoprecipitation, Incubation, Real-time Polymerase Chain Reaction

    Depletion of CRL7 SMU1 complex proteins induce mitotic defects. (A) HeLa cells were transfected with either control or SMU1 siRNAs. Cells were stained with antibody against α-tubulin to check the spindle defects (multipolar spindles) and nuclei were counterstained with DAPI to check for chromosomal defects (lagging chromosomes and anaphase bridges). Scale bar: 10 μm. (B) Quantification of results shown in A ( n =50 cells each). (C) HeLa cells were transduced with control shRNA, CUL7 shRNA, DDB1 shRNA or RNF40 shRNA. Cells were stained with antibody against α-tubulin to check the spindle defects (multipolar spindles) and nuclei were counterstained with DAPI to check for chromosomal defects (lagging chromosomes and anaphase bridges). Scale bar: 5 μm. (D) Quantification of results shown in C ( n =40 cells each). (E) Cells were transfected with H2B wild type (WT) and H2B K120R mutant. (F) Various mitotic abnormalities in cells expressing H2B WT and H2B K120R mutant were checked using immunofluorescence after staining with antibody against α-tubulin and DAPI. Scale bar: 5 μm. (G) Quantification of results shown in F ( n =50 cells each). Error bars indicate the mean+s.d.; *** P

    Journal: Journal of Cell Science

    Article Title: CRL7SMU1 E3 ligase complex-driven H2B ubiquitylation functions in sister chromatid cohesion by regulating SMC1 expression

    doi: 10.1242/jcs.213868

    Figure Lengend Snippet: Depletion of CRL7 SMU1 complex proteins induce mitotic defects. (A) HeLa cells were transfected with either control or SMU1 siRNAs. Cells were stained with antibody against α-tubulin to check the spindle defects (multipolar spindles) and nuclei were counterstained with DAPI to check for chromosomal defects (lagging chromosomes and anaphase bridges). Scale bar: 10 μm. (B) Quantification of results shown in A ( n =50 cells each). (C) HeLa cells were transduced with control shRNA, CUL7 shRNA, DDB1 shRNA or RNF40 shRNA. Cells were stained with antibody against α-tubulin to check the spindle defects (multipolar spindles) and nuclei were counterstained with DAPI to check for chromosomal defects (lagging chromosomes and anaphase bridges). Scale bar: 5 μm. (D) Quantification of results shown in C ( n =40 cells each). (E) Cells were transfected with H2B wild type (WT) and H2B K120R mutant. (F) Various mitotic abnormalities in cells expressing H2B WT and H2B K120R mutant were checked using immunofluorescence after staining with antibody against α-tubulin and DAPI. Scale bar: 5 μm. (G) Quantification of results shown in F ( n =50 cells each). Error bars indicate the mean+s.d.; *** P

    Article Snippet: Antibodies against SMU1 (Abgent #AT3965a; 1:1000); RNF40 (Sigma #R9029; 1:2000); CUL7 (Sigma #C1743; 1:2000); DDB1 (Bethyl #A300-462A; 1:5000); SMC1a (Abcam #ab133643; 1:1000); H2B (Millipore #07-371; 1:5000); histone H2B ubiquitylated at Lys120 (H2Bub; Cell Signaling Technology #5546; 1:1000); cyclin A (BD #611269; 1:1000); CDT1 (Bethyl #A300-786A; 1:1000); histone H3 phosphorylated at Ser10 (pH3; Cell Signaling Technology #9701L; for western blotting 1:1000, for immunofluorescence 1:200); Cul4a (Bethyl #A300-739A; 1:5000); RNF20 (Abcam #ab32629; 1:1000); Myc-tag (Santa Cruz #9E10; 1:1000); FLAG-tag (Sigma, #F3165; 1:10,000); HA (Bethyl, #A190-108A; 1:1000), actin (Sigma, #A5441; 1:10,000) and α-tubulin (Sigma, #T6074; for western blotting 1:5000, for immunofluorescence 1:200) were used in this study.

    Techniques: Transfection, Staining, Transduction, shRNA, Mutagenesis, Expressing, Immunofluorescence

    CRL7 SMU1 complex is necessary for driving SMC1 gene expression. (A) Exponentially growing HeLa cells were subjected to ChIP analysis using either anti-SMU1 or anti-IgG antibody. SMU1 enrichment at various loci is shown. The data shown is derived from three independent experiments. (B) Cells expressing control shRNA, CUL7 shRNA, DDB1 shRNA, RNF40 shRNA and SMU1 shRNA were subjected to ChIP analysis using H2Bub antibody. Fold change of H2Bub enrichment at indicated loci with respect to control shRNA is shown. The data shown is derived from three independent experiments. (C) Total RNA was extracted from HeLa cells transfected with control or SMU1 siRNA or CUL7 shRNA or DDB1 shRNA and RNF40 shRNA, and expression levels of various genes measured by qRT-PCR from three independent experiments is shown. (D) HeLa cells transduced with control, CUL7 shRNA, DDB1 shRNA, RNF40 shRNA or SMU1 shRNA and levels of SMC1a protein was measured by immunoblotting with specific antibody. (E) HeLa cells were transfected with H2B wild type (WT) and H2B K120R mutant. Relative expression of indicated genes measured by using qRT-PCR from three independent experiments was plotted. Error bars indicate the mean+s.d; *** P

    Journal: Journal of Cell Science

    Article Title: CRL7SMU1 E3 ligase complex-driven H2B ubiquitylation functions in sister chromatid cohesion by regulating SMC1 expression

    doi: 10.1242/jcs.213868

    Figure Lengend Snippet: CRL7 SMU1 complex is necessary for driving SMC1 gene expression. (A) Exponentially growing HeLa cells were subjected to ChIP analysis using either anti-SMU1 or anti-IgG antibody. SMU1 enrichment at various loci is shown. The data shown is derived from three independent experiments. (B) Cells expressing control shRNA, CUL7 shRNA, DDB1 shRNA, RNF40 shRNA and SMU1 shRNA were subjected to ChIP analysis using H2Bub antibody. Fold change of H2Bub enrichment at indicated loci with respect to control shRNA is shown. The data shown is derived from three independent experiments. (C) Total RNA was extracted from HeLa cells transfected with control or SMU1 siRNA or CUL7 shRNA or DDB1 shRNA and RNF40 shRNA, and expression levels of various genes measured by qRT-PCR from three independent experiments is shown. (D) HeLa cells transduced with control, CUL7 shRNA, DDB1 shRNA, RNF40 shRNA or SMU1 shRNA and levels of SMC1a protein was measured by immunoblotting with specific antibody. (E) HeLa cells were transfected with H2B wild type (WT) and H2B K120R mutant. Relative expression of indicated genes measured by using qRT-PCR from three independent experiments was plotted. Error bars indicate the mean+s.d; *** P

    Article Snippet: Antibodies against SMU1 (Abgent #AT3965a; 1:1000); RNF40 (Sigma #R9029; 1:2000); CUL7 (Sigma #C1743; 1:2000); DDB1 (Bethyl #A300-462A; 1:5000); SMC1a (Abcam #ab133643; 1:1000); H2B (Millipore #07-371; 1:5000); histone H2B ubiquitylated at Lys120 (H2Bub; Cell Signaling Technology #5546; 1:1000); cyclin A (BD #611269; 1:1000); CDT1 (Bethyl #A300-786A; 1:1000); histone H3 phosphorylated at Ser10 (pH3; Cell Signaling Technology #9701L; for western blotting 1:1000, for immunofluorescence 1:200); Cul4a (Bethyl #A300-739A; 1:5000); RNF20 (Abcam #ab32629; 1:1000); Myc-tag (Santa Cruz #9E10; 1:1000); FLAG-tag (Sigma, #F3165; 1:10,000); HA (Bethyl, #A190-108A; 1:1000), actin (Sigma, #A5441; 1:10,000) and α-tubulin (Sigma, #T6074; for western blotting 1:5000, for immunofluorescence 1:200) were used in this study.

    Techniques: Expressing, Chromatin Immunoprecipitation, Derivative Assay, shRNA, Transfection, Quantitative RT-PCR, Transduction, Mutagenesis

    CRL7 SMU1 complex regulates the monoubiquitylation of H2B at position K120. (A) SFB-tagged CUL7, DDB1, RNF40, SMU1, Rab7 or empty vector (EV) were transfected and interaction of H2B was detected by immunoblotting with specific antibody after streptavidin Sepharose pull-down. (B) HeLa cells were transduced with either control or SMU1-specific shRNA followed by overexpression of SFB-tagged RNF40. 72 h post transduction, pull-down was performed with streptavidin Sepharose beads, and interaction of DDB1 and H2B with RNF40 was evaluated by immunoblotting with their respective antibodies. (C) SFB-tagged SMU1 was overexpressed in cells transduced with either control or RNF40-specific shRNA. The interaction of SMU1 with H2B and DDB1 was detected through immunoblotting using specific antibodies after immunoprecipitation. (D,E) Cells were transduced with either control or DDB1 shRNA (E), and control or CUL7 shRNA containing viral particles. Pull-down followed by detection of different indicated proteins in precipitates were done as described in B. (F) Model shows the assembly of CRL7 SMU1 complex in association with its substrate H2B. (G) GST pull-down assay was performed with immobilized control GST or GST–SMU1 fusion proteins on glutathione beads, followed by incubation with bacterially purified MBP-H2B. The interaction of SMU1 with H2B was assessed by immunoblotting with anti-MBP antibody. Expression of GST, recombinant GST-SMU1 and MBP-H2B was shown by Coomassie Blue staining. (H) HeLa cells were transduced using either control or RNF40 shRNA. Post 72 h, cells were collected and lysed to isolate soluble and histone fractions. Lysates were subjected to SDS-PAGE followed by immunoblotting using the indicated antibodies. (I) Cells were transfected/transduced with either control or SMU1 siRNA, (J) or DDB1 shRNA, (K) or CUL7 shRNA. Soluble and acid-extracted histone fractions were subjected to SDS-PAGE followed by immunoblotting using indicated antibodies. The data presented here represent three independent experiments.

    Journal: Journal of Cell Science

    Article Title: CRL7SMU1 E3 ligase complex-driven H2B ubiquitylation functions in sister chromatid cohesion by regulating SMC1 expression

    doi: 10.1242/jcs.213868

    Figure Lengend Snippet: CRL7 SMU1 complex regulates the monoubiquitylation of H2B at position K120. (A) SFB-tagged CUL7, DDB1, RNF40, SMU1, Rab7 or empty vector (EV) were transfected and interaction of H2B was detected by immunoblotting with specific antibody after streptavidin Sepharose pull-down. (B) HeLa cells were transduced with either control or SMU1-specific shRNA followed by overexpression of SFB-tagged RNF40. 72 h post transduction, pull-down was performed with streptavidin Sepharose beads, and interaction of DDB1 and H2B with RNF40 was evaluated by immunoblotting with their respective antibodies. (C) SFB-tagged SMU1 was overexpressed in cells transduced with either control or RNF40-specific shRNA. The interaction of SMU1 with H2B and DDB1 was detected through immunoblotting using specific antibodies after immunoprecipitation. (D,E) Cells were transduced with either control or DDB1 shRNA (E), and control or CUL7 shRNA containing viral particles. Pull-down followed by detection of different indicated proteins in precipitates were done as described in B. (F) Model shows the assembly of CRL7 SMU1 complex in association with its substrate H2B. (G) GST pull-down assay was performed with immobilized control GST or GST–SMU1 fusion proteins on glutathione beads, followed by incubation with bacterially purified MBP-H2B. The interaction of SMU1 with H2B was assessed by immunoblotting with anti-MBP antibody. Expression of GST, recombinant GST-SMU1 and MBP-H2B was shown by Coomassie Blue staining. (H) HeLa cells were transduced using either control or RNF40 shRNA. Post 72 h, cells were collected and lysed to isolate soluble and histone fractions. Lysates were subjected to SDS-PAGE followed by immunoblotting using the indicated antibodies. (I) Cells were transfected/transduced with either control or SMU1 siRNA, (J) or DDB1 shRNA, (K) or CUL7 shRNA. Soluble and acid-extracted histone fractions were subjected to SDS-PAGE followed by immunoblotting using indicated antibodies. The data presented here represent three independent experiments.

    Article Snippet: Antibodies against SMU1 (Abgent #AT3965a; 1:1000); RNF40 (Sigma #R9029; 1:2000); CUL7 (Sigma #C1743; 1:2000); DDB1 (Bethyl #A300-462A; 1:5000); SMC1a (Abcam #ab133643; 1:1000); H2B (Millipore #07-371; 1:5000); histone H2B ubiquitylated at Lys120 (H2Bub; Cell Signaling Technology #5546; 1:1000); cyclin A (BD #611269; 1:1000); CDT1 (Bethyl #A300-786A; 1:1000); histone H3 phosphorylated at Ser10 (pH3; Cell Signaling Technology #9701L; for western blotting 1:1000, for immunofluorescence 1:200); Cul4a (Bethyl #A300-739A; 1:5000); RNF20 (Abcam #ab32629; 1:1000); Myc-tag (Santa Cruz #9E10; 1:1000); FLAG-tag (Sigma, #F3165; 1:10,000); HA (Bethyl, #A190-108A; 1:1000), actin (Sigma, #A5441; 1:10,000) and α-tubulin (Sigma, #T6074; for western blotting 1:5000, for immunofluorescence 1:200) were used in this study.

    Techniques: Plasmid Preparation, Transfection, Transduction, shRNA, Over Expression, Immunoprecipitation, Pull Down Assay, Incubation, Purification, Expressing, Recombinant, Staining, SDS Page

    Quantitative ChIP analyses of barrier-associated protein occupancy in the ankyrin-1 5′HS region. Quantitative ChIP studies were performed to examine barrier-associated protein occupancy in the ankyrin 5′HS region using K562 cell (left) and primary erythroid cell (right) chromatin. ( A ) was included as a negative control. ( B ) Methyltransferases. ChIP analyses of the ankyrin 5′HS region using antibodies against PRMT1 and PRMT4. A region of the CITED2 ) was included as a positive control, and a region of the hsSat2 was included as a negative control. ( C ) Histone acetylases and chromatin remodeling proteins. ChIP analyses of the ankyrin 5′HS region using antibodies against CBP, PCAF, and SMARCA4/BRG1. A region of the IL4 ), and a region of the hsSat2 was included as a negative control (–C) for all 3 antibodies. ( D ) The region and primers utilized in ChIP.

    Journal: The Journal of Clinical Investigation

    Article Title: Mutation of a barrier insulator in the human ankyrin-1 gene is associated with hereditary spherocytosis

    doi: 10.1172/JCI42240

    Figure Lengend Snippet: Quantitative ChIP analyses of barrier-associated protein occupancy in the ankyrin-1 5′HS region. Quantitative ChIP studies were performed to examine barrier-associated protein occupancy in the ankyrin 5′HS region using K562 cell (left) and primary erythroid cell (right) chromatin. ( A ) was included as a negative control. ( B ) Methyltransferases. ChIP analyses of the ankyrin 5′HS region using antibodies against PRMT1 and PRMT4. A region of the CITED2 ) was included as a positive control, and a region of the hsSat2 was included as a negative control. ( C ) Histone acetylases and chromatin remodeling proteins. ChIP analyses of the ankyrin 5′HS region using antibodies against CBP, PCAF, and SMARCA4/BRG1. A region of the IL4 ), and a region of the hsSat2 was included as a negative control (–C) for all 3 antibodies. ( D ) The region and primers utilized in ChIP.

    Article Snippet: Antibodies used for immunoprecipitation were diacetylated histone H3 (Upstate Biotechnology, 06-599), tetraacetylated histone H4 (Upstate Biotechnology, 06-866), CBP (Santa Cruz Biotechnology Inc., A-22), histone H3 trimethylated lysine 27 (Abcam, ab6002), histone H3 trimethylated lysine 9 (Abcam, ab8893), histone H3 dimethylated lysine 4 (Abcam, ab7766), P300 (Santa Cruz Biotechnology Inc., C-20, SC-585X), USF1 (Santa Cruz Biotechnology Inc., H-86, sc-8983), USF2 (Santa Cruz Biotechnology Inc., C-20, sc-862), and Brg1/SMARCA4 (Santa Cruz Biotechnology Inc., H-88, SC-10768X).

    Techniques: Chromatin Immunoprecipitation, Negative Control, Positive Control

    Accumulation in N. benthamiana Plants. (A) Downregulating expression of TFIIIA as well as 5S rRNA , shown by RNA gel blotting. Immunoblots of leaf extracts showed reduced levels of TFIIIA-7ZF as well as increased level of TFIIIA-9ZF upon antisense suppression of TFIIIA . Histone H3 served as a loading control for protein gel blots, while ethidium bromide staining of rRNAs served as a loading control for RNA gel blots. (B) upon ectopic expression of TFIIIA-7ZF but not of TFIIIA-9ZF . .

    Journal: The Plant Cell

    Article Title: A Land Plant-Specific Transcription Factor Directly Enhances Transcription of a Pathogenic Noncoding RNA Template by DNA-Dependent RNA Polymerase II [OPEN]

    doi: 10.1105/tpc.16.00100

    Figure Lengend Snippet: Accumulation in N. benthamiana Plants. (A) Downregulating expression of TFIIIA as well as 5S rRNA , shown by RNA gel blotting. Immunoblots of leaf extracts showed reduced levels of TFIIIA-7ZF as well as increased level of TFIIIA-9ZF upon antisense suppression of TFIIIA . Histone H3 served as a loading control for protein gel blots, while ethidium bromide staining of rRNAs served as a loading control for RNA gel blots. (B) upon ectopic expression of TFIIIA-7ZF but not of TFIIIA-9ZF . .

    Article Snippet: We used the following antibodies in our studies: 8WG16 (GeneTex) at 1:1000, anti-Histone H3 (Genscript) at 1:1000, anti-TFIIIA (described above) at 1:400, anti-FLAG L5 monoclonal antibody (Life Technologies) at 1:1000, horseradish peroxidase ( )-conjugated anti-HA (Sigma-Aldrich) at 1:1000 dilution, -conjugated anti-mouse IgG (Sigma-Aldrich) at 1:8000 dilution, -conjugated anti-rat IgG (Sigma-Aldrich) at 1:4000 dilution, and -conjugated protein A (Life Technologies) at 1:1000 dilution. (Note: NaF was omitted from all buffers when extracting Pol II for immunoblotting.)

    Techniques: Expressing, Western Blot, Staining