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Active Motif histone extraction
Histone Extraction, supplied by Active Motif, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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histone extraction - by Bioz Stars, 2021-07
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Related Articles

Methylation:

Article Title: Global Transcriptome Analysis Reveals That Poly(ADP-Ribose) Polymerase 1 Regulates Gene Expression through EZH2
Article Snippet: Purified histones were quantified using the Pierce BCA protein assay kit. .. H3K27me3 levels were quantified using a histone H3 methylated Lys27 enzyme-linked immunosorbent assay (ELISA) kit (Active Motif) according to the manufacturer's protocol. .. Briefly, histone extracts were added to the histone H3 antibody-coated plate, and the plate was incubated for 1 h at room temperature with agitation on a platform rocker.

Enzyme-linked Immunosorbent Assay:

Article Title: Global Transcriptome Analysis Reveals That Poly(ADP-Ribose) Polymerase 1 Regulates Gene Expression through EZH2
Article Snippet: Purified histones were quantified using the Pierce BCA protein assay kit. .. H3K27me3 levels were quantified using a histone H3 methylated Lys27 enzyme-linked immunosorbent assay (ELISA) kit (Active Motif) according to the manufacturer's protocol. .. Briefly, histone extracts were added to the histone H3 antibody-coated plate, and the plate was incubated for 1 h at room temperature with agitation on a platform rocker.

other:

Article Title: H3K23me2 is a new heterochromatic mark in Caenorhabditis elegans
Article Snippet: IF analyses performed in embryos showed that H3K23me2 is a dynamic mark, being reduced in PGCs (P4 and Z2/Z3), in comparison to somatic cells.

Western Blot:

Article Title: H3K56me3 Is a Novel, Conserved Heterochromatic Mark That Largely but Not Completely Overlaps with H3K9me3 in Both Regulation and Localization
Article Snippet: It will be of interest to see if H3K56me3 has an evolutionary conserved role in germline development, although its functional implication might be different in different metazoans. .. Supporting Information Immunoblot peptide competition experiments to determine specificity of αH3K9me3 antibodies used in this study. αH3K9me3 antibodies from (A) Active Motif or (B) the Jenuwein laboratory were pre-incubated with 2 µg/ml competitor peptides before addition to immunoblots containing recombinant H3 protein (R) or acid extracted HeLa Kyoto histones (H) (top). .. Ponceau staining (bottom) serves as loading control. (TIF) Click here for additional data file.

Recombinant:

Article Title: H3K56me3 Is a Novel, Conserved Heterochromatic Mark That Largely but Not Completely Overlaps with H3K9me3 in Both Regulation and Localization
Article Snippet: It will be of interest to see if H3K56me3 has an evolutionary conserved role in germline development, although its functional implication might be different in different metazoans. .. Supporting Information Immunoblot peptide competition experiments to determine specificity of αH3K9me3 antibodies used in this study. αH3K9me3 antibodies from (A) Active Motif or (B) the Jenuwein laboratory were pre-incubated with 2 µg/ml competitor peptides before addition to immunoblots containing recombinant H3 protein (R) or acid extracted HeLa Kyoto histones (H) (top). .. Ponceau staining (bottom) serves as loading control. (TIF) Click here for additional data file.

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  • 86
    Active Motif hdac deacetylase activity
    The NCOR complex interacts with HDAC3 to suppress myogenic differentiation in ERMS. ( A and B ) MF20 IF images of 381T cells with safe-harbor control CRISPR targeting ( A ) and NCOR1 and NCOR2 double-gene targeting ( B ). ( C ) Summary of MF20 IF in 381T cells with HDAC3 , NCOR1 , and NCOR2 CRISPR targeting. ( D ) Cell growth by cell counts normalized to cells with safe-harbor control targeting. ( E ) Schematics of CRISPR mutant HDAC3 (HDAC3m) expression constructs. ( F ) Assay of <t>HDAC</t> <t>deacetylase</t> activity in HDAC3m mutants at day 4 after HDAC3 knockout. ( G ) Western blot/co-IP assays assessing the interaction of HDAC3m mutants. ( H – O ) MF20 IF images and quantification data of HDAC3 CRISPR-inducible 381T cells transduced with empty-vector control ( H and I ), WT HDAC3m ( J and K ), or HDAC3m mutants ( L – O ) with or without tamoxifen (Tam) treatment. Error bars in C and D represent the mean ± SD of three biological replicates. All cell-culture experiments (viral transduction or tamoxifen treatment) were analyzed 10 d posttreatment. (Scale bars in B and H : 50 μm.) ** P
    Hdac Deacetylase Activity, supplied by Active Motif, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hdac deacetylase activity/product/Active Motif
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hdac deacetylase activity - by Bioz Stars, 2021-07
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    99
    Active Motif anti human h2a z
    Probing <t>H2A.Z</t> dynamics using VivosX. ( A ) HTZ1(T46C) FL strains containing the TBP-FRB or SWC5-FRB alleles or the corresponding wild-type alleles ( no FRB ) in the Anchor-away genetic background (W303 MAT a tor1-1 fpr1 RPL13A-2xFKBP12 ) ( Haruki et al., 2008 ) were incubated with rapamycin (+RAP) or without (i.e. DMSO, –RAP) for 1 hr before each culture was divided into halves, where one half was oxidized with 4-DPS (for 20 min) and the other without. Fixation, protein extraction, and immunoblotting analysis were conducted as described for Figure 1D . Bottom panels : Quantification of the ZxZ bands and the mono Z bands was performed as described in Figure 1D . ( B ) VivosX was performed using HTZ1(T46C) FL TBP-FRB yeast transformed with the [ V5 HTA1(N39C)-HTB1] plasmid. Rep: biological replicates. ( C ) Quantification of ( B ). The immunoblot signals of ZxZ, AxZ, and mono Z were normalized to total H2A.Z. Bars and error bars represent the means and standard deviations of three biological replicates. One asterisk (*) indicates p≤0.05 and two (**) indicates p≤0.01 of t-tests. 10.7554/eLife.36654.014 Values used to plot Figure 5A . 10.7554/eLife.36654.015 Values used to plot Figure 5C .
    Anti Human H2a Z, supplied by Active Motif, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti human h2a z/product/Active Motif
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti human h2a z - by Bioz Stars, 2021-07
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    86
    Active Motif anti h2a
    Probing <t>H2A.Z</t> dynamics using VivosX. ( A ) HTZ1(T46C) FL strains containing the TBP-FRB or SWC5-FRB alleles or the corresponding wild-type alleles ( no FRB ) in the Anchor-away genetic background (W303 MAT a tor1-1 fpr1 RPL13A-2xFKBP12 ) ( Haruki et al., 2008 ) were incubated with rapamycin (+RAP) or without (i.e. DMSO, –RAP) for 1 hr before each culture was divided into halves, where one half was oxidized with 4-DPS (for 20 min) and the other without. Fixation, protein extraction, and immunoblotting analysis were conducted as described for Figure 1D . Bottom panels : Quantification of the ZxZ bands and the mono Z bands was performed as described in Figure 1D . ( B ) VivosX was performed using HTZ1(T46C) FL TBP-FRB yeast transformed with the [ V5 HTA1(N39C)-HTB1] plasmid. Rep: biological replicates. ( C ) Quantification of ( B ). The immunoblot signals of ZxZ, AxZ, and mono Z were normalized to total H2A.Z. Bars and error bars represent the means and standard deviations of three biological replicates. One asterisk (*) indicates p≤0.05 and two (**) indicates p≤0.01 of t-tests. 10.7554/eLife.36654.014 Values used to plot Figure 5A . 10.7554/eLife.36654.015 Values used to plot Figure 5C .
    Anti H2a, supplied by Active Motif, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The NCOR complex interacts with HDAC3 to suppress myogenic differentiation in ERMS. ( A and B ) MF20 IF images of 381T cells with safe-harbor control CRISPR targeting ( A ) and NCOR1 and NCOR2 double-gene targeting ( B ). ( C ) Summary of MF20 IF in 381T cells with HDAC3 , NCOR1 , and NCOR2 CRISPR targeting. ( D ) Cell growth by cell counts normalized to cells with safe-harbor control targeting. ( E ) Schematics of CRISPR mutant HDAC3 (HDAC3m) expression constructs. ( F ) Assay of HDAC deacetylase activity in HDAC3m mutants at day 4 after HDAC3 knockout. ( G ) Western blot/co-IP assays assessing the interaction of HDAC3m mutants. ( H – O ) MF20 IF images and quantification data of HDAC3 CRISPR-inducible 381T cells transduced with empty-vector control ( H and I ), WT HDAC3m ( J and K ), or HDAC3m mutants ( L – O ) with or without tamoxifen (Tam) treatment. Error bars in C and D represent the mean ± SD of three biological replicates. All cell-culture experiments (viral transduction or tamoxifen treatment) were analyzed 10 d posttreatment. (Scale bars in B and H : 50 μm.) ** P

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

    Article Title: CRISPR screen identifies the NCOR/HDAC3 complex as a major suppressor of differentiation in rhabdomyosarcoma

    doi: 10.1073/pnas.1610270114

    Figure Lengend Snippet: The NCOR complex interacts with HDAC3 to suppress myogenic differentiation in ERMS. ( A and B ) MF20 IF images of 381T cells with safe-harbor control CRISPR targeting ( A ) and NCOR1 and NCOR2 double-gene targeting ( B ). ( C ) Summary of MF20 IF in 381T cells with HDAC3 , NCOR1 , and NCOR2 CRISPR targeting. ( D ) Cell growth by cell counts normalized to cells with safe-harbor control targeting. ( E ) Schematics of CRISPR mutant HDAC3 (HDAC3m) expression constructs. ( F ) Assay of HDAC deacetylase activity in HDAC3m mutants at day 4 after HDAC3 knockout. ( G ) Western blot/co-IP assays assessing the interaction of HDAC3m mutants. ( H – O ) MF20 IF images and quantification data of HDAC3 CRISPR-inducible 381T cells transduced with empty-vector control ( H and I ), WT HDAC3m ( J and K ), or HDAC3m mutants ( L – O ) with or without tamoxifen (Tam) treatment. Error bars in C and D represent the mean ± SD of three biological replicates. All cell-culture experiments (viral transduction or tamoxifen treatment) were analyzed 10 d posttreatment. (Scale bars in B and H : 50 μm.) ** P

    Article Snippet: HDAC3 (WT or mutant) was immunoprecipitated from nuclear extracts, and HDAC deacetylase activity was quantified using a fluorescent HDAC activity assay kit (Active Motif, catalog no. 56200), which uses a short peptide substrate that contains an acetylated lysine residue that can be deacetylated by class I, IIB, and IV HDAC enzymes.

    Techniques: CRISPR, Mutagenesis, Expressing, Construct, Histone Deacetylase Assay, Activity Assay, Knock-Out, Western Blot, Co-Immunoprecipitation Assay, Transduction, Plasmid Preparation, Cell Culture

    Probing H2A.Z dynamics using VivosX. ( A ) HTZ1(T46C) FL strains containing the TBP-FRB or SWC5-FRB alleles or the corresponding wild-type alleles ( no FRB ) in the Anchor-away genetic background (W303 MAT a tor1-1 fpr1 RPL13A-2xFKBP12 ) ( Haruki et al., 2008 ) were incubated with rapamycin (+RAP) or without (i.e. DMSO, –RAP) for 1 hr before each culture was divided into halves, where one half was oxidized with 4-DPS (for 20 min) and the other without. Fixation, protein extraction, and immunoblotting analysis were conducted as described for Figure 1D . Bottom panels : Quantification of the ZxZ bands and the mono Z bands was performed as described in Figure 1D . ( B ) VivosX was performed using HTZ1(T46C) FL TBP-FRB yeast transformed with the [ V5 HTA1(N39C)-HTB1] plasmid. Rep: biological replicates. ( C ) Quantification of ( B ). The immunoblot signals of ZxZ, AxZ, and mono Z were normalized to total H2A.Z. Bars and error bars represent the means and standard deviations of three biological replicates. One asterisk (*) indicates p≤0.05 and two (**) indicates p≤0.01 of t-tests. 10.7554/eLife.36654.014 Values used to plot Figure 5A . 10.7554/eLife.36654.015 Values used to plot Figure 5C .

    Journal: eLife

    Article Title: VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

    doi: 10.7554/eLife.36654

    Figure Lengend Snippet: Probing H2A.Z dynamics using VivosX. ( A ) HTZ1(T46C) FL strains containing the TBP-FRB or SWC5-FRB alleles or the corresponding wild-type alleles ( no FRB ) in the Anchor-away genetic background (W303 MAT a tor1-1 fpr1 RPL13A-2xFKBP12 ) ( Haruki et al., 2008 ) were incubated with rapamycin (+RAP) or without (i.e. DMSO, –RAP) for 1 hr before each culture was divided into halves, where one half was oxidized with 4-DPS (for 20 min) and the other without. Fixation, protein extraction, and immunoblotting analysis were conducted as described for Figure 1D . Bottom panels : Quantification of the ZxZ bands and the mono Z bands was performed as described in Figure 1D . ( B ) VivosX was performed using HTZ1(T46C) FL TBP-FRB yeast transformed with the [ V5 HTA1(N39C)-HTB1] plasmid. Rep: biological replicates. ( C ) Quantification of ( B ). The immunoblot signals of ZxZ, AxZ, and mono Z were normalized to total H2A.Z. Bars and error bars represent the means and standard deviations of three biological replicates. One asterisk (*) indicates p≤0.05 and two (**) indicates p≤0.01 of t-tests. 10.7554/eLife.36654.014 Values used to plot Figure 5A . 10.7554/eLife.36654.015 Values used to plot Figure 5C .

    Article Snippet: Anti-FLAG (Sigma Aldrich , Cat. F3165) was used at a dilution of 1:2000, anti-H2A (Active Motif, Cat. 39235) at 1:2000, anti-H3 (gift of Carl Wu) at 1:2000, anti-V5 (Thermo Fisher , Cat. MA5-15253) at 1:2000, and anti-human H2A.Z (Active Motif, Cat. 39943) at 1:2000.

    Techniques: Incubation, Protein Extraction, Transformation Assay, Plasmid Preparation

    Domain analysis of Swc5 using H2A.Z VivosX. ( A ) The cartoon depicts the domain organization of yeast Swc5 ( Sun and Luk, 2017 ). ( B,C ) The yeast strain, HTZ1(T46C) FL swc5∆ , was transformed by a single-copy plasmid containing either the wild-type SWC5 or the indicated SWC5 mutants or by the control vector ( swc5∆ ). Top panels: Each strain was represented by three independent transformants and analyzed by VivosX in parallel. Htz1(T46C) FL and its crosslinked (ZxZ) adducts was detected by anti-FLAG immunoblotting. Bottom panels: Quantification of the immunoblots above. Bars and error bars represent the means and standard deviations of three biological replicates.

    Journal: eLife

    Article Title: VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

    doi: 10.7554/eLife.36654

    Figure Lengend Snippet: Domain analysis of Swc5 using H2A.Z VivosX. ( A ) The cartoon depicts the domain organization of yeast Swc5 ( Sun and Luk, 2017 ). ( B,C ) The yeast strain, HTZ1(T46C) FL swc5∆ , was transformed by a single-copy plasmid containing either the wild-type SWC5 or the indicated SWC5 mutants or by the control vector ( swc5∆ ). Top panels: Each strain was represented by three independent transformants and analyzed by VivosX in parallel. Htz1(T46C) FL and its crosslinked (ZxZ) adducts was detected by anti-FLAG immunoblotting. Bottom panels: Quantification of the immunoblots above. Bars and error bars represent the means and standard deviations of three biological replicates.

    Article Snippet: Anti-FLAG (Sigma Aldrich , Cat. F3165) was used at a dilution of 1:2000, anti-H2A (Active Motif, Cat. 39235) at 1:2000, anti-H3 (gift of Carl Wu) at 1:2000, anti-V5 (Thermo Fisher , Cat. MA5-15253) at 1:2000, and anti-human H2A.Z (Active Motif, Cat. 39943) at 1:2000.

    Techniques: Transformation Assay, Plasmid Preparation, Western Blot

    Control experiments for yeast VivosX. ( A,B ) Immunoblots showing the different effects of the FLAG and V5 epitope tags on the SDS-PAGE mobility of the H2A.Z and H2A histones. ( C ) DNA samples purified from chromatin isolated from the indicated strains before and after MNase digestion were analyzed by agarose gel electrophoresis and SYBR Green staining. 1x MNase represents 0.08 U/µL of MNase. Circled lanes: MNase digestion conditions selected for the experiment in Figure 3D . ( D,E ) Logarithmically growing HTZ1(T46C) FL cells were fixed with sodium azide (0.1%) for 15 min on ice before treatment with and without 4-DPS (180 µM) for 20 min. The cells were then fixed with TCA fixation and the proteins extracted and analyzed as described for Figure 1D .

    Journal: eLife

    Article Title: VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

    doi: 10.7554/eLife.36654

    Figure Lengend Snippet: Control experiments for yeast VivosX. ( A,B ) Immunoblots showing the different effects of the FLAG and V5 epitope tags on the SDS-PAGE mobility of the H2A.Z and H2A histones. ( C ) DNA samples purified from chromatin isolated from the indicated strains before and after MNase digestion were analyzed by agarose gel electrophoresis and SYBR Green staining. 1x MNase represents 0.08 U/µL of MNase. Circled lanes: MNase digestion conditions selected for the experiment in Figure 3D . ( D,E ) Logarithmically growing HTZ1(T46C) FL cells were fixed with sodium azide (0.1%) for 15 min on ice before treatment with and without 4-DPS (180 µM) for 20 min. The cells were then fixed with TCA fixation and the proteins extracted and analyzed as described for Figure 1D .

    Article Snippet: Anti-FLAG (Sigma Aldrich , Cat. F3165) was used at a dilution of 1:2000, anti-H2A (Active Motif, Cat. 39235) at 1:2000, anti-H3 (gift of Carl Wu) at 1:2000, anti-V5 (Thermo Fisher , Cat. MA5-15253) at 1:2000, and anti-human H2A.Z (Active Motif, Cat. 39943) at 1:2000.

    Techniques: Western Blot, SDS Page, Purification, Isolation, Agarose Gel Electrophoresis, SYBR Green Assay, Staining

    Estimation of ectopic H2A.Z level. ( A ) Total lysates of MCF10A cells transfected with H2AFZ(H46C) V5 or H2AFZ (untagged) driven by the same tetracycline-inducible promoter were analyzed by immunoblotting using an antibody directed against the C-terminus of human H2A.Z. ( B ) The same blot was probed with the anti-V5 antibody. Note that the H2A.Z antibody failed to detect the V5-tagged H2A.Z because the V5 tag at the C-terminus interfered with antibody recognition. In addition, the ubiquitylation sites of H2A.Z overlap the epitope of the anti-H2A.Z antibody. This may explain the disproportionately low H2A.Z-Ub signal in the anti-H2A.Z blot in comparison to the anti-V5 blot. The H2A.Z-Ub recognition bias by the C-terminus H2A.Z antibodies was previously observed ( Sarcinella et al., 2007 ).

    Journal: eLife

    Article Title: VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

    doi: 10.7554/eLife.36654

    Figure Lengend Snippet: Estimation of ectopic H2A.Z level. ( A ) Total lysates of MCF10A cells transfected with H2AFZ(H46C) V5 or H2AFZ (untagged) driven by the same tetracycline-inducible promoter were analyzed by immunoblotting using an antibody directed against the C-terminus of human H2A.Z. ( B ) The same blot was probed with the anti-V5 antibody. Note that the H2A.Z antibody failed to detect the V5-tagged H2A.Z because the V5 tag at the C-terminus interfered with antibody recognition. In addition, the ubiquitylation sites of H2A.Z overlap the epitope of the anti-H2A.Z antibody. This may explain the disproportionately low H2A.Z-Ub signal in the anti-H2A.Z blot in comparison to the anti-V5 blot. The H2A.Z-Ub recognition bias by the C-terminus H2A.Z antibodies was previously observed ( Sarcinella et al., 2007 ).

    Article Snippet: Anti-FLAG (Sigma Aldrich , Cat. F3165) was used at a dilution of 1:2000, anti-H2A (Active Motif, Cat. 39235) at 1:2000, anti-H3 (gift of Carl Wu) at 1:2000, anti-V5 (Thermo Fisher , Cat. MA5-15253) at 1:2000, and anti-human H2A.Z (Active Motif, Cat. 39943) at 1:2000.

    Techniques: Transfection

    The thiol-disulfide interchange reaction between the cysteine thiols and 4-DPS. ( A ) A proposed mechanism of the thiol-disulfide interchange reaction between cysteine thiols and 4-DPS. Light and dark green loops represent the L1 regions of the opposite nucleosomal H2A.Z (or H2A) pair at the L1-L1’ interface. ( B ) The proposed clamshell opening of the nucleosome structure could facilitate the conformational dynamics of the L1-L1’ interface. Dotted arrows: direction of movement.

    Journal: eLife

    Article Title: VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

    doi: 10.7554/eLife.36654

    Figure Lengend Snippet: The thiol-disulfide interchange reaction between the cysteine thiols and 4-DPS. ( A ) A proposed mechanism of the thiol-disulfide interchange reaction between cysteine thiols and 4-DPS. Light and dark green loops represent the L1 regions of the opposite nucleosomal H2A.Z (or H2A) pair at the L1-L1’ interface. ( B ) The proposed clamshell opening of the nucleosome structure could facilitate the conformational dynamics of the L1-L1’ interface. Dotted arrows: direction of movement.

    Article Snippet: Anti-FLAG (Sigma Aldrich , Cat. F3165) was used at a dilution of 1:2000, anti-H2A (Active Motif, Cat. 39235) at 1:2000, anti-H3 (gift of Carl Wu) at 1:2000, anti-V5 (Thermo Fisher , Cat. MA5-15253) at 1:2000, and anti-human H2A.Z (Active Motif, Cat. 39943) at 1:2000.

    Techniques:

    Relative Cα-Cα’ distances at the L1-L1’ interface of AA and ZZ nucleosomes. ( A,B ) Cartoons depicting the anti-parallel L1 and L1’ loops of yeast H2A.Z and H2A. ( C ) A table of Cα-Cα’ distances measured between the indicated amino acid positions in the AA and ZZ nucleosomes. The Cα-Cα’ distances for H2A.Z are based on the published mouse/frog ZZ nucleosome structure ( Suto et al., 2000 ) but the corresponding positions of yeast Htz1 were indicated. The Cα-Cα’ distances are based on the yeast AA nucleosome structure ( White et al., 2001 ).

    Journal: eLife

    Article Title: VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

    doi: 10.7554/eLife.36654

    Figure Lengend Snippet: Relative Cα-Cα’ distances at the L1-L1’ interface of AA and ZZ nucleosomes. ( A,B ) Cartoons depicting the anti-parallel L1 and L1’ loops of yeast H2A.Z and H2A. ( C ) A table of Cα-Cα’ distances measured between the indicated amino acid positions in the AA and ZZ nucleosomes. The Cα-Cα’ distances for H2A.Z are based on the published mouse/frog ZZ nucleosome structure ( Suto et al., 2000 ) but the corresponding positions of yeast Htz1 were indicated. The Cα-Cα’ distances are based on the yeast AA nucleosome structure ( White et al., 2001 ).

    Article Snippet: Anti-FLAG (Sigma Aldrich , Cat. F3165) was used at a dilution of 1:2000, anti-H2A (Active Motif, Cat. 39235) at 1:2000, anti-H3 (gift of Carl Wu) at 1:2000, anti-V5 (Thermo Fisher , Cat. MA5-15253) at 1:2000, and anti-human H2A.Z (Active Motif, Cat. 39943) at 1:2000.

    Techniques:

    Altered expression of MAPK-related genes and cyclins in response to FGF-2 correlates with H3K27ac levels. A) The signal intensity of H3K27ac, normalized to input, is plotted over the regions surrounding unique TSS positions for the FBS and FGF conditions after 5 hrs of treatment. Replicates were merged for plotting. TSS regions are clustered into 4 groups (G1-4) by k-means based on signal intensity. B) The change in gene expression between the FGF and FBS conditions at 5 hrs, as assessed by RNA-seq, is plotted against the corresponding change in H3K27ac intensity. Each point represents a single gene (9,248 in total) and peaks were required to be within 10 kb of the TSS. For genes with multiple peaks, the peak closest to the TSS (and then the most intense peak in the case of ties) was considered. Contour plots are based only on changes in H3K27ac (q

    Journal: bioRxiv

    Article Title: PROFILING OF Y1 CELLS TREATED WITH FGF-2 REVEALS PARALLELS WITH ONCOGENE-INDUCED SENESCENCE

    doi: 10.1101/2020.08.17.247023

    Figure Lengend Snippet: Altered expression of MAPK-related genes and cyclins in response to FGF-2 correlates with H3K27ac levels. A) The signal intensity of H3K27ac, normalized to input, is plotted over the regions surrounding unique TSS positions for the FBS and FGF conditions after 5 hrs of treatment. Replicates were merged for plotting. TSS regions are clustered into 4 groups (G1-4) by k-means based on signal intensity. B) The change in gene expression between the FGF and FBS conditions at 5 hrs, as assessed by RNA-seq, is plotted against the corresponding change in H3K27ac intensity. Each point represents a single gene (9,248 in total) and peaks were required to be within 10 kb of the TSS. For genes with multiple peaks, the peak closest to the TSS (and then the most intense peak in the case of ties) was considered. Contour plots are based only on changes in H3K27ac (q

    Article Snippet: Extracts were incubated overnight at 4°C with 5 μg of anti-H3K27ac (Active Motif) prebound to protein G Dynabeads (Thermo Scientific) that were previously blocked in PBS with 0.5% BSA.

    Techniques: Expressing, RNA Sequencing Assay

    Gene ontology analysis of H3K27ac peaks with differential abundance after FGF-2 treatment. A,B) H3K27ac peaks with quantitative differences between the FGF treatment and the FBS control were annotated with the nearest gene. GO enrichment analysis was performed for peaks with lower intensity in the FGF condition (A, 502 peaks representing 415 genes, log2(FGF/FBS)

    Journal: bioRxiv

    Article Title: PROFILING OF Y1 CELLS TREATED WITH FGF-2 REVEALS PARALLELS WITH ONCOGENE-INDUCED SENESCENCE

    doi: 10.1101/2020.08.17.247023

    Figure Lengend Snippet: Gene ontology analysis of H3K27ac peaks with differential abundance after FGF-2 treatment. A,B) H3K27ac peaks with quantitative differences between the FGF treatment and the FBS control were annotated with the nearest gene. GO enrichment analysis was performed for peaks with lower intensity in the FGF condition (A, 502 peaks representing 415 genes, log2(FGF/FBS)

    Article Snippet: Extracts were incubated overnight at 4°C with 5 μg of anti-H3K27ac (Active Motif) prebound to protein G Dynabeads (Thermo Scientific) that were previously blocked in PBS with 0.5% BSA.

    Techniques:

    Summary of H3K27ac ChIP peaks and their genomic locations. A) After alignment and processing, H3K27ac ChIP-seq data was analyzed in R with the DiffBind ( Stark Brown, 2011 ), ChIPseeker ( Yu et al , 2015 ), and ClusterProfiler ( Yu et al , 2012 ) packages. Principal component analysis (PCA) was used to cluster the samples based on 40,783 consensus H3K27ac peaks. Each point represents an individual replicate, labeled as 1 or 2, from the FBS or FGF condition after 5 hrs of treatment. B) PCA was also performed considering only the 3,246 differential H3K27ac peaks (q

    Journal: bioRxiv

    Article Title: PROFILING OF Y1 CELLS TREATED WITH FGF-2 REVEALS PARALLELS WITH ONCOGENE-INDUCED SENESCENCE

    doi: 10.1101/2020.08.17.247023

    Figure Lengend Snippet: Summary of H3K27ac ChIP peaks and their genomic locations. A) After alignment and processing, H3K27ac ChIP-seq data was analyzed in R with the DiffBind ( Stark Brown, 2011 ), ChIPseeker ( Yu et al , 2015 ), and ClusterProfiler ( Yu et al , 2012 ) packages. Principal component analysis (PCA) was used to cluster the samples based on 40,783 consensus H3K27ac peaks. Each point represents an individual replicate, labeled as 1 or 2, from the FBS or FGF condition after 5 hrs of treatment. B) PCA was also performed considering only the 3,246 differential H3K27ac peaks (q

    Article Snippet: Extracts were incubated overnight at 4°C with 5 μg of anti-H3K27ac (Active Motif) prebound to protein G Dynabeads (Thermo Scientific) that were previously blocked in PBS with 0.5% BSA.

    Techniques: Chromatin Immunoprecipitation, Labeling

    Gene ontology analysis of genes with matching trends in levels of RNA and H3K27ac. A,B) Gene ontology analysis was performed on genes showing concordant trends in RNA and H3K27ac levels. Enriched terms are presented for (A) genes with increased RNA expression and H3K27ac intensity (157 genes with H3K27ac log2(FGF/FBS) > 0 at FDR

    Journal: bioRxiv

    Article Title: PROFILING OF Y1 CELLS TREATED WITH FGF-2 REVEALS PARALLELS WITH ONCOGENE-INDUCED SENESCENCE

    doi: 10.1101/2020.08.17.247023

    Figure Lengend Snippet: Gene ontology analysis of genes with matching trends in levels of RNA and H3K27ac. A,B) Gene ontology analysis was performed on genes showing concordant trends in RNA and H3K27ac levels. Enriched terms are presented for (A) genes with increased RNA expression and H3K27ac intensity (157 genes with H3K27ac log2(FGF/FBS) > 0 at FDR

    Article Snippet: Extracts were incubated overnight at 4°C with 5 μg of anti-H3K27ac (Active Motif) prebound to protein G Dynabeads (Thermo Scientific) that were previously blocked in PBS with 0.5% BSA.

    Techniques: RNA Expression

    Probing H2A.Z dynamics using VivosX. ( A ) HTZ1(T46C) FL strains containing the TBP-FRB or SWC5-FRB alleles or the corresponding wild-type alleles ( no FRB ) in the Anchor-away genetic background (W303 MAT a tor1-1 fpr1 RPL13A-2xFKBP12 ) ( Haruki et al., 2008 ) were incubated with rapamycin (+RAP) or without (i.e. DMSO, –RAP) for 1 hr before each culture was divided into halves, where one half was oxidized with 4-DPS (for 20 min) and the other without. Fixation, protein extraction, and immunoblotting analysis were conducted as described for Figure 1D . Bottom panels : Quantification of the ZxZ bands and the mono Z bands was performed as described in Figure 1D . ( B ) VivosX was performed using HTZ1(T46C) FL TBP-FRB yeast transformed with the [ V5 HTA1(N39C)-HTB1] plasmid. Rep: biological replicates. ( C ) Quantification of ( B ). The immunoblot signals of ZxZ, AxZ, and mono Z were normalized to total H2A.Z. Bars and error bars represent the means and standard deviations of three biological replicates. One asterisk (*) indicates p≤0.05 and two (**) indicates p≤0.01 of t-tests. 10.7554/eLife.36654.014 Values used to plot Figure 5A . 10.7554/eLife.36654.015 Values used to plot Figure 5C .

    Journal: eLife

    Article Title: VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

    doi: 10.7554/eLife.36654

    Figure Lengend Snippet: Probing H2A.Z dynamics using VivosX. ( A ) HTZ1(T46C) FL strains containing the TBP-FRB or SWC5-FRB alleles or the corresponding wild-type alleles ( no FRB ) in the Anchor-away genetic background (W303 MAT a tor1-1 fpr1 RPL13A-2xFKBP12 ) ( Haruki et al., 2008 ) were incubated with rapamycin (+RAP) or without (i.e. DMSO, –RAP) for 1 hr before each culture was divided into halves, where one half was oxidized with 4-DPS (for 20 min) and the other without. Fixation, protein extraction, and immunoblotting analysis were conducted as described for Figure 1D . Bottom panels : Quantification of the ZxZ bands and the mono Z bands was performed as described in Figure 1D . ( B ) VivosX was performed using HTZ1(T46C) FL TBP-FRB yeast transformed with the [ V5 HTA1(N39C)-HTB1] plasmid. Rep: biological replicates. ( C ) Quantification of ( B ). The immunoblot signals of ZxZ, AxZ, and mono Z were normalized to total H2A.Z. Bars and error bars represent the means and standard deviations of three biological replicates. One asterisk (*) indicates p≤0.05 and two (**) indicates p≤0.01 of t-tests. 10.7554/eLife.36654.014 Values used to plot Figure 5A . 10.7554/eLife.36654.015 Values used to plot Figure 5C .

    Article Snippet: Anti-FLAG (Sigma Aldrich , Cat. F3165) was used at a dilution of 1:2000, anti-H2A (Active Motif, Cat. 39235) at 1:2000, anti-H3 (gift of Carl Wu) at 1:2000, anti-V5 (Thermo Fisher , Cat. MA5-15253) at 1:2000, and anti-human H2A.Z (Active Motif, Cat. 39943) at 1:2000.

    Techniques: Incubation, Protein Extraction, Transformation Assay, Plasmid Preparation

    Cysteine substitution at multiple positions of the L1 region of H2A supports A-to-A crosslinking in yeast cells. ( A ) The cartoon depicts the plasmid shuffle yeast system used to verify the functionality of the V5 HTA1(Cys) mutants. ( B ) The ability of the HTA1(Cys) mutants to complement the lack of endogenous genes for H2A and H2B was indicated by growth in the presence of 5-FOA, which removes the wild type [ HTA1-HTB1-URA3] vector from the cells. ( C,D ) VivosX analysis of yeast H2A was performed as described in Figure 1D except that the immunoblots were probed with an anti-H2A antibody ( Active Motif ). Asterisk (*) indicates a non-specific band. ( E ) Quantification of the AxA adducts and the uncrosslinked H2A (mono A) was performed as described in Figure 1F . Bars and error bars indicate the means and standard deviation of three biological replicates. 10.7554/eLife.36654.009 Values used to plot Figure 2E .

    Journal: eLife

    Article Title: VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

    doi: 10.7554/eLife.36654

    Figure Lengend Snippet: Cysteine substitution at multiple positions of the L1 region of H2A supports A-to-A crosslinking in yeast cells. ( A ) The cartoon depicts the plasmid shuffle yeast system used to verify the functionality of the V5 HTA1(Cys) mutants. ( B ) The ability of the HTA1(Cys) mutants to complement the lack of endogenous genes for H2A and H2B was indicated by growth in the presence of 5-FOA, which removes the wild type [ HTA1-HTB1-URA3] vector from the cells. ( C,D ) VivosX analysis of yeast H2A was performed as described in Figure 1D except that the immunoblots were probed with an anti-H2A antibody ( Active Motif ). Asterisk (*) indicates a non-specific band. ( E ) Quantification of the AxA adducts and the uncrosslinked H2A (mono A) was performed as described in Figure 1F . Bars and error bars indicate the means and standard deviation of three biological replicates. 10.7554/eLife.36654.009 Values used to plot Figure 2E .

    Article Snippet: Anti-FLAG (Sigma Aldrich , Cat. F3165) was used at a dilution of 1:2000, anti-H2A (Active Motif, Cat. 39235) at 1:2000, anti-H3 (gift of Carl Wu) at 1:2000, anti-V5 (Thermo Fisher , Cat. MA5-15253) at 1:2000, and anti-human H2A.Z (Active Motif, Cat. 39943) at 1:2000.

    Techniques: Plasmid Preparation, Western Blot, Standard Deviation

    Domain analysis of Swc5 using H2A.Z VivosX. ( A ) The cartoon depicts the domain organization of yeast Swc5 ( Sun and Luk, 2017 ). ( B,C ) The yeast strain, HTZ1(T46C) FL swc5∆ , was transformed by a single-copy plasmid containing either the wild-type SWC5 or the indicated SWC5 mutants or by the control vector ( swc5∆ ). Top panels: Each strain was represented by three independent transformants and analyzed by VivosX in parallel. Htz1(T46C) FL and its crosslinked (ZxZ) adducts was detected by anti-FLAG immunoblotting. Bottom panels: Quantification of the immunoblots above. Bars and error bars represent the means and standard deviations of three biological replicates.

    Journal: eLife

    Article Title: VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

    doi: 10.7554/eLife.36654

    Figure Lengend Snippet: Domain analysis of Swc5 using H2A.Z VivosX. ( A ) The cartoon depicts the domain organization of yeast Swc5 ( Sun and Luk, 2017 ). ( B,C ) The yeast strain, HTZ1(T46C) FL swc5∆ , was transformed by a single-copy plasmid containing either the wild-type SWC5 or the indicated SWC5 mutants or by the control vector ( swc5∆ ). Top panels: Each strain was represented by three independent transformants and analyzed by VivosX in parallel. Htz1(T46C) FL and its crosslinked (ZxZ) adducts was detected by anti-FLAG immunoblotting. Bottom panels: Quantification of the immunoblots above. Bars and error bars represent the means and standard deviations of three biological replicates.

    Article Snippet: Anti-FLAG (Sigma Aldrich , Cat. F3165) was used at a dilution of 1:2000, anti-H2A (Active Motif, Cat. 39235) at 1:2000, anti-H3 (gift of Carl Wu) at 1:2000, anti-V5 (Thermo Fisher , Cat. MA5-15253) at 1:2000, and anti-human H2A.Z (Active Motif, Cat. 39943) at 1:2000.

    Techniques: Transformation Assay, Plasmid Preparation, Western Blot

    Control experiments for yeast VivosX. ( A,B ) Immunoblots showing the different effects of the FLAG and V5 epitope tags on the SDS-PAGE mobility of the H2A.Z and H2A histones. ( C ) DNA samples purified from chromatin isolated from the indicated strains before and after MNase digestion were analyzed by agarose gel electrophoresis and SYBR Green staining. 1x MNase represents 0.08 U/µL of MNase. Circled lanes: MNase digestion conditions selected for the experiment in Figure 3D . ( D,E ) Logarithmically growing HTZ1(T46C) FL cells were fixed with sodium azide (0.1%) for 15 min on ice before treatment with and without 4-DPS (180 µM) for 20 min. The cells were then fixed with TCA fixation and the proteins extracted and analyzed as described for Figure 1D .

    Journal: eLife

    Article Title: VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

    doi: 10.7554/eLife.36654

    Figure Lengend Snippet: Control experiments for yeast VivosX. ( A,B ) Immunoblots showing the different effects of the FLAG and V5 epitope tags on the SDS-PAGE mobility of the H2A.Z and H2A histones. ( C ) DNA samples purified from chromatin isolated from the indicated strains before and after MNase digestion were analyzed by agarose gel electrophoresis and SYBR Green staining. 1x MNase represents 0.08 U/µL of MNase. Circled lanes: MNase digestion conditions selected for the experiment in Figure 3D . ( D,E ) Logarithmically growing HTZ1(T46C) FL cells were fixed with sodium azide (0.1%) for 15 min on ice before treatment with and without 4-DPS (180 µM) for 20 min. The cells were then fixed with TCA fixation and the proteins extracted and analyzed as described for Figure 1D .

    Article Snippet: Anti-FLAG (Sigma Aldrich , Cat. F3165) was used at a dilution of 1:2000, anti-H2A (Active Motif, Cat. 39235) at 1:2000, anti-H3 (gift of Carl Wu) at 1:2000, anti-V5 (Thermo Fisher , Cat. MA5-15253) at 1:2000, and anti-human H2A.Z (Active Motif, Cat. 39943) at 1:2000.

    Techniques: Western Blot, SDS Page, Purification, Isolation, Agarose Gel Electrophoresis, SYBR Green Assay, Staining

    A cartoon depicting the proposed histone cycle. Z-B dimers are in green, A-B dimers in red and H3-H4 tetramers in gray. NFR: Nucleosome free region. Black arrows: transcription start site. +1: the nucleosome immediately downstream of a promoter. PIC: preinitiation complex. Balls in cyan: cysteine probes. Cysteine probes on H2A are not shown for simplicity.

    Journal: eLife

    Article Title: VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

    doi: 10.7554/eLife.36654

    Figure Lengend Snippet: A cartoon depicting the proposed histone cycle. Z-B dimers are in green, A-B dimers in red and H3-H4 tetramers in gray. NFR: Nucleosome free region. Black arrows: transcription start site. +1: the nucleosome immediately downstream of a promoter. PIC: preinitiation complex. Balls in cyan: cysteine probes. Cysteine probes on H2A are not shown for simplicity.

    Article Snippet: Anti-FLAG (Sigma Aldrich , Cat. F3165) was used at a dilution of 1:2000, anti-H2A (Active Motif, Cat. 39235) at 1:2000, anti-H3 (gift of Carl Wu) at 1:2000, anti-V5 (Thermo Fisher , Cat. MA5-15253) at 1:2000, and anti-human H2A.Z (Active Motif, Cat. 39943) at 1:2000.

    Techniques:

    Estimation of ectopic H2A.Z level. ( A ) Total lysates of MCF10A cells transfected with H2AFZ(H46C) V5 or H2AFZ (untagged) driven by the same tetracycline-inducible promoter were analyzed by immunoblotting using an antibody directed against the C-terminus of human H2A.Z. ( B ) The same blot was probed with the anti-V5 antibody. Note that the H2A.Z antibody failed to detect the V5-tagged H2A.Z because the V5 tag at the C-terminus interfered with antibody recognition. In addition, the ubiquitylation sites of H2A.Z overlap the epitope of the anti-H2A.Z antibody. This may explain the disproportionately low H2A.Z-Ub signal in the anti-H2A.Z blot in comparison to the anti-V5 blot. The H2A.Z-Ub recognition bias by the C-terminus H2A.Z antibodies was previously observed ( Sarcinella et al., 2007 ).

    Journal: eLife

    Article Title: VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

    doi: 10.7554/eLife.36654

    Figure Lengend Snippet: Estimation of ectopic H2A.Z level. ( A ) Total lysates of MCF10A cells transfected with H2AFZ(H46C) V5 or H2AFZ (untagged) driven by the same tetracycline-inducible promoter were analyzed by immunoblotting using an antibody directed against the C-terminus of human H2A.Z. ( B ) The same blot was probed with the anti-V5 antibody. Note that the H2A.Z antibody failed to detect the V5-tagged H2A.Z because the V5 tag at the C-terminus interfered with antibody recognition. In addition, the ubiquitylation sites of H2A.Z overlap the epitope of the anti-H2A.Z antibody. This may explain the disproportionately low H2A.Z-Ub signal in the anti-H2A.Z blot in comparison to the anti-V5 blot. The H2A.Z-Ub recognition bias by the C-terminus H2A.Z antibodies was previously observed ( Sarcinella et al., 2007 ).

    Article Snippet: Anti-FLAG (Sigma Aldrich , Cat. F3165) was used at a dilution of 1:2000, anti-H2A (Active Motif, Cat. 39235) at 1:2000, anti-H3 (gift of Carl Wu) at 1:2000, anti-V5 (Thermo Fisher , Cat. MA5-15253) at 1:2000, and anti-human H2A.Z (Active Motif, Cat. 39943) at 1:2000.

    Techniques: Transfection

    The thiol-disulfide interchange reaction between the cysteine thiols and 4-DPS. ( A ) A proposed mechanism of the thiol-disulfide interchange reaction between cysteine thiols and 4-DPS. Light and dark green loops represent the L1 regions of the opposite nucleosomal H2A.Z (or H2A) pair at the L1-L1’ interface. ( B ) The proposed clamshell opening of the nucleosome structure could facilitate the conformational dynamics of the L1-L1’ interface. Dotted arrows: direction of movement.

    Journal: eLife

    Article Title: VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

    doi: 10.7554/eLife.36654

    Figure Lengend Snippet: The thiol-disulfide interchange reaction between the cysteine thiols and 4-DPS. ( A ) A proposed mechanism of the thiol-disulfide interchange reaction between cysteine thiols and 4-DPS. Light and dark green loops represent the L1 regions of the opposite nucleosomal H2A.Z (or H2A) pair at the L1-L1’ interface. ( B ) The proposed clamshell opening of the nucleosome structure could facilitate the conformational dynamics of the L1-L1’ interface. Dotted arrows: direction of movement.

    Article Snippet: Anti-FLAG (Sigma Aldrich , Cat. F3165) was used at a dilution of 1:2000, anti-H2A (Active Motif, Cat. 39235) at 1:2000, anti-H3 (gift of Carl Wu) at 1:2000, anti-V5 (Thermo Fisher , Cat. MA5-15253) at 1:2000, and anti-human H2A.Z (Active Motif, Cat. 39943) at 1:2000.

    Techniques:

    Relative Cα-Cα’ distances at the L1-L1’ interface of AA and ZZ nucleosomes. ( A,B ) Cartoons depicting the anti-parallel L1 and L1’ loops of yeast H2A.Z and H2A. ( C ) A table of Cα-Cα’ distances measured between the indicated amino acid positions in the AA and ZZ nucleosomes. The Cα-Cα’ distances for H2A.Z are based on the published mouse/frog ZZ nucleosome structure ( Suto et al., 2000 ) but the corresponding positions of yeast Htz1 were indicated. The Cα-Cα’ distances are based on the yeast AA nucleosome structure ( White et al., 2001 ).

    Journal: eLife

    Article Title: VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

    doi: 10.7554/eLife.36654

    Figure Lengend Snippet: Relative Cα-Cα’ distances at the L1-L1’ interface of AA and ZZ nucleosomes. ( A,B ) Cartoons depicting the anti-parallel L1 and L1’ loops of yeast H2A.Z and H2A. ( C ) A table of Cα-Cα’ distances measured between the indicated amino acid positions in the AA and ZZ nucleosomes. The Cα-Cα’ distances for H2A.Z are based on the published mouse/frog ZZ nucleosome structure ( Suto et al., 2000 ) but the corresponding positions of yeast Htz1 were indicated. The Cα-Cα’ distances are based on the yeast AA nucleosome structure ( White et al., 2001 ).

    Article Snippet: Anti-FLAG (Sigma Aldrich , Cat. F3165) was used at a dilution of 1:2000, anti-H2A (Active Motif, Cat. 39235) at 1:2000, anti-H3 (gift of Carl Wu) at 1:2000, anti-V5 (Thermo Fisher , Cat. MA5-15253) at 1:2000, and anti-human H2A.Z (Active Motif, Cat. 39943) at 1:2000.

    Techniques: