abcam histone h3  (Abcam)

 
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    Histone H3 acetyl K9 Assay Kit In Situ
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    Abcam abcam histone h3

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    Average 93 stars, based on 2 article reviews
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    Article Title: Histone H3K4 and K36 Methylation, Chd1 and Rpd3S Oppose the Functions of Saccharomyces cerevisiae Spt4-Spt5 in Transcription
    Article Snippet: Interestingly, deletion of the N-terminal tail of histone H3 gave a strong His+ phenotype.

    Article Title: A Nucleosome Surface Formed by Histone H4, H2A, and H3 Residues Is Needed for Proper Histone H3 Lys36 Methylation, Histone Acetylation, and Repression of Cryptic Transcription *
    Article Snippet: In addition, histone H3, H2A, and Set2 proteins levels are similar in WT and all of the histone mutants examined ( A ).

    Article Title: Specialized compartments of cardiac nuclei exhibit distinct proteomic anatomy *
    Article Snippet: As expected histone H3 and H2B were exclusively identified in chromatin and further enriched in acid extracted fractions, whereas the soluble spliceosomal factor, U170K, was restricted to the nucleoplasm.

    Mass Spectrometry:

    Article Title: The Histone Deacetylase Inhibitor, MS-275 (Entinostat), Downregulates c-FLIP, Sensitizes Osteosarcoma Cells to FasL, and Induces the Regression of Osteosarcoma Lung Metastases
    Article Snippet: .. MS-275 induced a time-dependent increase in acetylated histone H3 which peaked at 48 hours, the timepoint at which MS-275 was able to both inhibit clonogenic survival and sensitize cells to sFasL. .. The total levels of histone H3 remained unaltered ( ).

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  • h2a z  (Abcam)
    93
    Abcam h2a z
    Acetylation of <t>H2A.Z</t> is required for upregulation of Hes1 Notch target gene. ( A ) Schematic representation of the Flag-RBP-J/Tip60 fusion proteins used in Figure 4B and C and in Supplementary Figures S9 and S10 . Amino acid numbering is accordingly to accession NP_033061.3 for RBP-J and NP_874368.1 for Tip60. RBP-J domain: LAG1-DNAbind, LAG1 DNA binding (CDD:255260); Tip60 domain: MOZ/SAS, MOZ/SAS family (CDD:250916). ( B ) RBP-J/Tip60 wildtype (wt) but not its catalytic dead (cd) mutant upregulates Hes1 expression in MT cells. MT cells were infected with retroviral particles delivering plasmids encoding Flag-tagged RBP-J/Tip60-wt, cd mutant or empty vector (Control). Total RNA was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp or Hes1 . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD ([**] P
    H2a Z, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Abcam eef2 fraction
    Co-immune precipitations reveal <t>eEF2</t> interactions with Dph6 and Dph5. (A) eEF2 interacts with Dph6 in a fashion that is independent of Dph7. (B) eEF2 interaction with Dph5 is dramatically enhanced by elimination of Dph7 or Dph1. Yeast strains co-expressing (His) 6 -tagged eEF2 with Dph6-HA (A) or Dph5-HA (B) in the background of wild-type (A: DPH7 and B: wt) and dph mutant strains (A: dph7 ; B: dph1 , dph6 and dph7 ) were subjected to immune precipitations (IP) using the anti-HA antibody. Strains expressing (His) 6 -tagged eEF2 on their own served as IP controls (A and B: no HA-tag). Subsequently, the precipitates were probed with anti-HA (A: top left panel; B: first panel) and anti-(His) 6 antibodies (A: bottom left panel) to check for the content of Dph6-HA (A) and Dph5-HA (B), respectively (all indicated by arrows). The content of HA-tagged Dph6 (A) and Dph5 (B) as well as (His) 6 -marked eEF2 (A and B) in the protein extracts prior to IP (pre-IP) was examined on individual Western blots using anti-HA (A: top right panel; B: fourth panel) and anti-(His) 6 antibodies (A: bottom right panel; B: third panel), respectively. While absence of Dph7 hardly affected the Dph6•eEF2 interaction (A), Dph5•eEF2 interaction was strongly enhanced by inactivating DPH7 or DPH1 (B).
    Eef2 Fraction, supplied by Abcam, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Acetylation of H2A.Z is required for upregulation of Hes1 Notch target gene. ( A ) Schematic representation of the Flag-RBP-J/Tip60 fusion proteins used in Figure 4B and C and in Supplementary Figures S9 and S10 . Amino acid numbering is accordingly to accession NP_033061.3 for RBP-J and NP_874368.1 for Tip60. RBP-J domain: LAG1-DNAbind, LAG1 DNA binding (CDD:255260); Tip60 domain: MOZ/SAS, MOZ/SAS family (CDD:250916). ( B ) RBP-J/Tip60 wildtype (wt) but not its catalytic dead (cd) mutant upregulates Hes1 expression in MT cells. MT cells were infected with retroviral particles delivering plasmids encoding Flag-tagged RBP-J/Tip60-wt, cd mutant or empty vector (Control). Total RNA was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp or Hes1 . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD ([**] P

    Journal: Nucleic Acids Research

    Article Title: Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response

    doi: 10.1093/nar/gky551

    Figure Lengend Snippet: Acetylation of H2A.Z is required for upregulation of Hes1 Notch target gene. ( A ) Schematic representation of the Flag-RBP-J/Tip60 fusion proteins used in Figure 4B and C and in Supplementary Figures S9 and S10 . Amino acid numbering is accordingly to accession NP_033061.3 for RBP-J and NP_874368.1 for Tip60. RBP-J domain: LAG1-DNAbind, LAG1 DNA binding (CDD:255260); Tip60 domain: MOZ/SAS, MOZ/SAS family (CDD:250916). ( B ) RBP-J/Tip60 wildtype (wt) but not its catalytic dead (cd) mutant upregulates Hes1 expression in MT cells. MT cells were infected with retroviral particles delivering plasmids encoding Flag-tagged RBP-J/Tip60-wt, cd mutant or empty vector (Control). Total RNA was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp or Hes1 . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD ([**] P

    Article Snippet: In order to evaluate whether Tip60 is responsible for the acetylation of H2A.Z in higher eukaryotes and to evaluate the transcriptional consequences of the Tip60-mediated acetylation of H2A.Z in the context of Notch signaling, we directly fused the transcription factor RBP-J to Tip60 wildtype (wt) or its catalytic-dead mutant [(cd, ( ) and Figure A].

    Techniques: Binding Assay, Mutagenesis, Expressing, Infection, Plasmid Preparation, Real-time Polymerase Chain Reaction

    Depletion of Drosophila His2Av/H2A.Z, dTip60 and domino/p400 leads to an asymmetric response to Delta-dependent Notch signaling and reveals a strong requirement for these factors in Notch responsive cells. ( A ) Drosophila homologs of RBP-J and p400 proteins, Su(H) and Domino respectively, physically interact with each other. GST pull-down experiments were performed using bacterially purified GST-Su(H) and in vitro transcribed/translated Domino aa 1557–2352 corresponding to the human RBP-J interacting p400–2 fragment (Figure 3D ). ( B ) Scheme of a Drosophila wing disc with the dorsal wing pouch highlighted in green. Notch activation by its ligands Delta/DLL and Serrate/Jagged at the dorsal (d)/ventral (v) boundary results in activation of target genes, such as wingless ( wg , in red; a: anterior, p: posterior, N act: activated Notch). Graph shows that the relative area of dorsal wing pouch is reduced in wing discs bearing clones of Delta -expressing cells that are depleted of His2Av/H2A.Z, dTip60 or domino/p400 . Data are mean ± SD ([**] P

    Journal: Nucleic Acids Research

    Article Title: Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response

    doi: 10.1093/nar/gky551

    Figure Lengend Snippet: Depletion of Drosophila His2Av/H2A.Z, dTip60 and domino/p400 leads to an asymmetric response to Delta-dependent Notch signaling and reveals a strong requirement for these factors in Notch responsive cells. ( A ) Drosophila homologs of RBP-J and p400 proteins, Su(H) and Domino respectively, physically interact with each other. GST pull-down experiments were performed using bacterially purified GST-Su(H) and in vitro transcribed/translated Domino aa 1557–2352 corresponding to the human RBP-J interacting p400–2 fragment (Figure 3D ). ( B ) Scheme of a Drosophila wing disc with the dorsal wing pouch highlighted in green. Notch activation by its ligands Delta/DLL and Serrate/Jagged at the dorsal (d)/ventral (v) boundary results in activation of target genes, such as wingless ( wg , in red; a: anterior, p: posterior, N act: activated Notch). Graph shows that the relative area of dorsal wing pouch is reduced in wing discs bearing clones of Delta -expressing cells that are depleted of His2Av/H2A.Z, dTip60 or domino/p400 . Data are mean ± SD ([**] P

    Article Snippet: In order to evaluate whether Tip60 is responsible for the acetylation of H2A.Z in higher eukaryotes and to evaluate the transcriptional consequences of the Tip60-mediated acetylation of H2A.Z in the context of Notch signaling, we directly fused the transcription factor RBP-J to Tip60 wildtype (wt) or its catalytic-dead mutant [(cd, ( ) and Figure A].

    Techniques: Purification, In Vitro, Activation Assay, Activated Clotting Time Assay, Clone Assay, Expressing

    H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. ( A ) Schematic representation of the NICD-inducible system established in MT cells. The NICD was fused to the estrogen receptor binding domain (NICD-ER) and retrovirally introduced into MT cells. The NICD-ER fusion protein is retained into the cytoplasm unless cells are treated with ( Z )-4-hydroxytamoxifen (4-OHT) that induces its nuclear translocation and activation of Notch target genes. ( B ) Hes1 and Il2ra Notch target genes are induced upon 4-OHT treatment of MT NICD-ER cells. Total RNA from MT NICD-ER cells, treated for 24 h with 4-OHT or EtOH as control, was reverse transcribed into cDNA and analyzed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of three independent experiments. ( C ) H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. MT NICD-ER cells were treated for 24 h with 4-OHT or EtOH as control and subjected to ChIP analysis using antibodies against H2A.Z, H2A.Zac, H3 or IgG as control. The qPCR analysis was focused at the Notch-dependent enhancers (red squares) represented on the left ( Hes1 +0.6 kb and Il2ra -26 kb ). Chrom X was used as negative control ( Control ). Data were normalized to the positive control ( GAPDH 0 kb ) and, in the case of H2A.Zac/H2A.Z, the H2A.Zac signals were further normalized to H2A.Z. Shown is the mean ± SD of two independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response

    doi: 10.1093/nar/gky551

    Figure Lengend Snippet: H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. ( A ) Schematic representation of the NICD-inducible system established in MT cells. The NICD was fused to the estrogen receptor binding domain (NICD-ER) and retrovirally introduced into MT cells. The NICD-ER fusion protein is retained into the cytoplasm unless cells are treated with ( Z )-4-hydroxytamoxifen (4-OHT) that induces its nuclear translocation and activation of Notch target genes. ( B ) Hes1 and Il2ra Notch target genes are induced upon 4-OHT treatment of MT NICD-ER cells. Total RNA from MT NICD-ER cells, treated for 24 h with 4-OHT or EtOH as control, was reverse transcribed into cDNA and analyzed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of three independent experiments. ( C ) H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. MT NICD-ER cells were treated for 24 h with 4-OHT or EtOH as control and subjected to ChIP analysis using antibodies against H2A.Z, H2A.Zac, H3 or IgG as control. The qPCR analysis was focused at the Notch-dependent enhancers (red squares) represented on the left ( Hes1 +0.6 kb and Il2ra -26 kb ). Chrom X was used as negative control ( Control ). Data were normalized to the positive control ( GAPDH 0 kb ) and, in the case of H2A.Zac/H2A.Z, the H2A.Zac signals were further normalized to H2A.Z. Shown is the mean ± SD of two independent experiments.

    Article Snippet: In order to evaluate whether Tip60 is responsible for the acetylation of H2A.Z in higher eukaryotes and to evaluate the transcriptional consequences of the Tip60-mediated acetylation of H2A.Z in the context of Notch signaling, we directly fused the transcription factor RBP-J to Tip60 wildtype (wt) or its catalytic-dead mutant [(cd, ( ) and Figure A].

    Techniques: Activation Assay, Binding Assay, Translocation Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control, Positive Control

    Histone variant H2A.Z has a negative impact on the expression of Notch target genes. ( A ) Histone Variant H2A.Z is efficiently depleted by CRISPR/Cas9 in MT cells. Whole Cell Extract (WCE) was prepared from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells and analysed by Western blotting. GAPDH was used as loading control. ( B ) Hes1 and Il2ra Notch target genes are upregulated upon depletion of H2A.Z. Total RNA from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of five independent experiments ([*] P

    Journal: Nucleic Acids Research

    Article Title: Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response

    doi: 10.1093/nar/gky551

    Figure Lengend Snippet: Histone variant H2A.Z has a negative impact on the expression of Notch target genes. ( A ) Histone Variant H2A.Z is efficiently depleted by CRISPR/Cas9 in MT cells. Whole Cell Extract (WCE) was prepared from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells and analysed by Western blotting. GAPDH was used as loading control. ( B ) Hes1 and Il2ra Notch target genes are upregulated upon depletion of H2A.Z. Total RNA from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of five independent experiments ([*] P

    Article Snippet: In order to evaluate whether Tip60 is responsible for the acetylation of H2A.Z in higher eukaryotes and to evaluate the transcriptional consequences of the Tip60-mediated acetylation of H2A.Z in the context of Notch signaling, we directly fused the transcription factor RBP-J to Tip60 wildtype (wt) or its catalytic-dead mutant [(cd, ( ) and Figure A].

    Techniques: Variant Assay, Expressing, CRISPR, Western Blot, Real-time Polymerase Chain Reaction

    Schematic summary representing the link between H2A.Z and Notch signaling. In absence of Notch signaling (OFF, left side), H2A.Z occupancy increases while its acetylation (H2A.Zac) is low. Upon Notch activation (ON, right side), H2A.Z occupancy is reduced while its acetylation increases.

    Journal: Nucleic Acids Research

    Article Title: Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response

    doi: 10.1093/nar/gky551

    Figure Lengend Snippet: Schematic summary representing the link between H2A.Z and Notch signaling. In absence of Notch signaling (OFF, left side), H2A.Z occupancy increases while its acetylation (H2A.Zac) is low. Upon Notch activation (ON, right side), H2A.Z occupancy is reduced while its acetylation increases.

    Article Snippet: In order to evaluate whether Tip60 is responsible for the acetylation of H2A.Z in higher eukaryotes and to evaluate the transcriptional consequences of the Tip60-mediated acetylation of H2A.Z in the context of Notch signaling, we directly fused the transcription factor RBP-J to Tip60 wildtype (wt) or its catalytic-dead mutant [(cd, ( ) and Figure A].

    Techniques: Activation Assay

    Extrahepatic transmission of epigenetic modifications and evidence for modifications in DNA methylation at fibrogenic regulator gene associated with liver disease progression in humans a–b) ChIP analysis of trimethylated H3 lysine 27 (H3K27me3) and histone variant H2A.Z enrichment at the rat PPAR-γ gene promoter in mature sperm collected from male adult rats that were given chronic CCl 4 or olive oil (control) for 4 weeks, then recovered for 2 weeks (n=5). All ChIP results in a) to e) are expressed as fold control isotype matched antibody. b) ChIP analysis as in a) was carried out on mature sperm isolated from male adult rats that underwent bile duct ligation (BDL) or sham operation (control) 15 days previously. c) ChIP analysis as in a) was carried out on mature sperm isolated from rats that received twice weekly intravenous serum transfers (for four weeks total) from control or rats that were given CCl 4 for 4 weeks and serum collected 48hrs following last injection (n=6) d) ChIP analysis as in a) was carried out on primary rat mesenchymal stem cells which were treated with control or 48hrs conditioned activated HSC media for 72hrs (n=3). e) ChIP analysis as in a) was carried out on human PPAR-γ gene promoter in primary human mesenchymal stem cells which were treated with quiescent (day 1) or activated HSC (day 15) conditioned media for 72hrs (n=3). f) DNA methylation at particular CG dinucleotides within human PPAR-γ promoter in NAFLD patients liver biopsy tissues was determined by pyrosequencing. Position of the differentially methylated CGs is shown in the schematic drawing above the graphs. Differences are expressed as percentage DNA methylation Statistical analysis; Mann Whitney test, where p=0.0013 for CpG1 and p=0.0047 for CpG2.

    Journal: Nature medicine

    Article Title: Multigenerational Epigenetic Adaptation of the Hepatic Wound-Healing Response

    doi: 10.1038/nm.2893

    Figure Lengend Snippet: Extrahepatic transmission of epigenetic modifications and evidence for modifications in DNA methylation at fibrogenic regulator gene associated with liver disease progression in humans a–b) ChIP analysis of trimethylated H3 lysine 27 (H3K27me3) and histone variant H2A.Z enrichment at the rat PPAR-γ gene promoter in mature sperm collected from male adult rats that were given chronic CCl 4 or olive oil (control) for 4 weeks, then recovered for 2 weeks (n=5). All ChIP results in a) to e) are expressed as fold control isotype matched antibody. b) ChIP analysis as in a) was carried out on mature sperm isolated from male adult rats that underwent bile duct ligation (BDL) or sham operation (control) 15 days previously. c) ChIP analysis as in a) was carried out on mature sperm isolated from rats that received twice weekly intravenous serum transfers (for four weeks total) from control or rats that were given CCl 4 for 4 weeks and serum collected 48hrs following last injection (n=6) d) ChIP analysis as in a) was carried out on primary rat mesenchymal stem cells which were treated with control or 48hrs conditioned activated HSC media for 72hrs (n=3). e) ChIP analysis as in a) was carried out on human PPAR-γ gene promoter in primary human mesenchymal stem cells which were treated with quiescent (day 1) or activated HSC (day 15) conditioned media for 72hrs (n=3). f) DNA methylation at particular CG dinucleotides within human PPAR-γ promoter in NAFLD patients liver biopsy tissues was determined by pyrosequencing. Position of the differentially methylated CGs is shown in the schematic drawing above the graphs. Differences are expressed as percentage DNA methylation Statistical analysis; Mann Whitney test, where p=0.0013 for CpG1 and p=0.0047 for CpG2.

    Article Snippet: Precleared chromatin was incubated with anti H2A.Z, anti H3K27me3 or isotype matched control antibody overnight at 4 degrees C. We added one hundred microliters of blocked Staph A membranes to ChIP reactions for two hours at 4 degrees C, then samples washed, eluted and genomic DNA purified as previously outlined .

    Techniques: Transmission Assay, DNA Methylation Assay, Chromatin Immunoprecipitation, Variant Assay, Isolation, Ligation, Injection, Methylation, MANN-WHITNEY

    Co-immune precipitations reveal eEF2 interactions with Dph6 and Dph5. (A) eEF2 interacts with Dph6 in a fashion that is independent of Dph7. (B) eEF2 interaction with Dph5 is dramatically enhanced by elimination of Dph7 or Dph1. Yeast strains co-expressing (His) 6 -tagged eEF2 with Dph6-HA (A) or Dph5-HA (B) in the background of wild-type (A: DPH7 and B: wt) and dph mutant strains (A: dph7 ; B: dph1 , dph6 and dph7 ) were subjected to immune precipitations (IP) using the anti-HA antibody. Strains expressing (His) 6 -tagged eEF2 on their own served as IP controls (A and B: no HA-tag). Subsequently, the precipitates were probed with anti-HA (A: top left panel; B: first panel) and anti-(His) 6 antibodies (A: bottom left panel) to check for the content of Dph6-HA (A) and Dph5-HA (B), respectively (all indicated by arrows). The content of HA-tagged Dph6 (A) and Dph5 (B) as well as (His) 6 -marked eEF2 (A and B) in the protein extracts prior to IP (pre-IP) was examined on individual Western blots using anti-HA (A: top right panel; B: fourth panel) and anti-(His) 6 antibodies (A: bottom right panel; B: third panel), respectively. While absence of Dph7 hardly affected the Dph6•eEF2 interaction (A), Dph5•eEF2 interaction was strongly enhanced by inactivating DPH7 or DPH1 (B).

    Journal: PLoS Genetics

    Article Title: The Amidation Step of Diphthamide Biosynthesis in Yeast Requires DPH6, a Gene Identified through Mining the DPH1-DPH5 Interaction Network

    doi: 10.1371/journal.pgen.1003334

    Figure Lengend Snippet: Co-immune precipitations reveal eEF2 interactions with Dph6 and Dph5. (A) eEF2 interacts with Dph6 in a fashion that is independent of Dph7. (B) eEF2 interaction with Dph5 is dramatically enhanced by elimination of Dph7 or Dph1. Yeast strains co-expressing (His) 6 -tagged eEF2 with Dph6-HA (A) or Dph5-HA (B) in the background of wild-type (A: DPH7 and B: wt) and dph mutant strains (A: dph7 ; B: dph1 , dph6 and dph7 ) were subjected to immune precipitations (IP) using the anti-HA antibody. Strains expressing (His) 6 -tagged eEF2 on their own served as IP controls (A and B: no HA-tag). Subsequently, the precipitates were probed with anti-HA (A: top left panel; B: first panel) and anti-(His) 6 antibodies (A: bottom left panel) to check for the content of Dph6-HA (A) and Dph5-HA (B), respectively (all indicated by arrows). The content of HA-tagged Dph6 (A) and Dph5 (B) as well as (His) 6 -marked eEF2 (A and B) in the protein extracts prior to IP (pre-IP) was examined on individual Western blots using anti-HA (A: top right panel; B: fourth panel) and anti-(His) 6 antibodies (A: bottom right panel; B: third panel), respectively. While absence of Dph7 hardly affected the Dph6•eEF2 interaction (A), Dph5•eEF2 interaction was strongly enhanced by inactivating DPH7 or DPH1 (B).

    Article Snippet: The identity of purified eEF2 fraction was confirmed by SDS-PAGE and Western blot analysis using an anti-(His)6 antibody (Abcam, #ab18184).

    Techniques: Expressing, Mutagenesis, Western Blot

    The biosynthetic pathway for modification of eEF2 by diphthamide. For roles played by the bona fide diphthamide genes DPH1–DPH5 in steps 1 and 2 of the pathway, see main text. The ill-defined step 3, conversion of diphthine to diphthamide by amidation, is highlighted (red label). It likely involves ATP and ammonium cofactors in a reaction catalyzed by unidentified DPH gene product(s). Step 4 indicates diphthamide can be hijacked for eEF2 inactivation and cell death induction by antifungals, i.e. sordarin and bacterial ADP ribosylase toxins (ADPRtox); alternatively, it has been reported to undergo cell growth related physiological ADP ribosylation (ADPRphys?) by elusive cellular modifier(s). ACP, 2-[3-amino-carboxyl-propyl]-histidine; SAM: S-adenosylmethionine.

    Journal: PLoS Genetics

    Article Title: The Amidation Step of Diphthamide Biosynthesis in Yeast Requires DPH6, a Gene Identified through Mining the DPH1-DPH5 Interaction Network

    doi: 10.1371/journal.pgen.1003334

    Figure Lengend Snippet: The biosynthetic pathway for modification of eEF2 by diphthamide. For roles played by the bona fide diphthamide genes DPH1–DPH5 in steps 1 and 2 of the pathway, see main text. The ill-defined step 3, conversion of diphthine to diphthamide by amidation, is highlighted (red label). It likely involves ATP and ammonium cofactors in a reaction catalyzed by unidentified DPH gene product(s). Step 4 indicates diphthamide can be hijacked for eEF2 inactivation and cell death induction by antifungals, i.e. sordarin and bacterial ADP ribosylase toxins (ADPRtox); alternatively, it has been reported to undergo cell growth related physiological ADP ribosylation (ADPRphys?) by elusive cellular modifier(s). ACP, 2-[3-amino-carboxyl-propyl]-histidine; SAM: S-adenosylmethionine.

    Article Snippet: The identity of purified eEF2 fraction was confirmed by SDS-PAGE and Western blot analysis using an anti-(His)6 antibody (Abcam, #ab18184).

    Techniques: Modification

    DPH6 and DPH7 deletion strains copy traits typically related to the bona fide diphthamide mutants dph1-dph5 . (A) Sordarin resistance. Ten-fold serial cell dilutions of the indicated yeast strains, BY4741 wild-type (wt) background and its dph1-dph7 gene deletion derivatives (upper panels) as well an MKK-derived eft1 eft2 double deletion background maintaining plasmid p EFT2 wild-type or H 699 substitution (H 699 N and H 699 I) alleles of EFT2 (lower panels), were grown on YPD plates in the absence (control) or presence (+sor) of 10 µg ml −1 sordarin. Growth was assayed for 3 d at 30°C. Sordarin resistant (R) and sensitive (S) responses are indicated. (B) Lack of in vitro ADP ribose acceptor activity of eEF2. Cell extracts obtained from dph1 , dph5 , dph6 and dph7 mutant and wild-type (wt) strains were incubated with (+DT) or without (−DT) 20 nM diphtheria toxin in the presence of biotin-NAD (10 µM) at 37°C for 1 hour. The transfer of biotin-ADP-ribose to eEF2 was detected by Western blotting using a streptavidin-conjugate. Two unknown non-specific bands (indicated by *) served as internal controls for even sample loading. (C) DT phenotype. As indicated, yeast dph mutants and wild-type control (wt) were tested for sensitivity to intracellular expression of DTA, the cytotoxic ADP ribosylase fragment of DT. This in vivo assay involved galactose-inducible expression from vector pSU8 (see Materials and Methods ). Serial cell dilutions were replica spotted onto raffinose (2% raf) and galactose-inducing conditions using concentrations (2, 0.2 and 0.1% gal) suited to achieve gradual down-regulation of DTA toxicity. Growth was for 3 days at 30°C. DTA sensitive (S) resistant (R), partially resistant (PR) and reduced sensitive (RS) phenotypes are indicated.

    Journal: PLoS Genetics

    Article Title: The Amidation Step of Diphthamide Biosynthesis in Yeast Requires DPH6, a Gene Identified through Mining the DPH1-DPH5 Interaction Network

    doi: 10.1371/journal.pgen.1003334

    Figure Lengend Snippet: DPH6 and DPH7 deletion strains copy traits typically related to the bona fide diphthamide mutants dph1-dph5 . (A) Sordarin resistance. Ten-fold serial cell dilutions of the indicated yeast strains, BY4741 wild-type (wt) background and its dph1-dph7 gene deletion derivatives (upper panels) as well an MKK-derived eft1 eft2 double deletion background maintaining plasmid p EFT2 wild-type or H 699 substitution (H 699 N and H 699 I) alleles of EFT2 (lower panels), were grown on YPD plates in the absence (control) or presence (+sor) of 10 µg ml −1 sordarin. Growth was assayed for 3 d at 30°C. Sordarin resistant (R) and sensitive (S) responses are indicated. (B) Lack of in vitro ADP ribose acceptor activity of eEF2. Cell extracts obtained from dph1 , dph5 , dph6 and dph7 mutant and wild-type (wt) strains were incubated with (+DT) or without (−DT) 20 nM diphtheria toxin in the presence of biotin-NAD (10 µM) at 37°C for 1 hour. The transfer of biotin-ADP-ribose to eEF2 was detected by Western blotting using a streptavidin-conjugate. Two unknown non-specific bands (indicated by *) served as internal controls for even sample loading. (C) DT phenotype. As indicated, yeast dph mutants and wild-type control (wt) were tested for sensitivity to intracellular expression of DTA, the cytotoxic ADP ribosylase fragment of DT. This in vivo assay involved galactose-inducible expression from vector pSU8 (see Materials and Methods ). Serial cell dilutions were replica spotted onto raffinose (2% raf) and galactose-inducing conditions using concentrations (2, 0.2 and 0.1% gal) suited to achieve gradual down-regulation of DTA toxicity. Growth was for 3 days at 30°C. DTA sensitive (S) resistant (R), partially resistant (PR) and reduced sensitive (RS) phenotypes are indicated.

    Article Snippet: The identity of purified eEF2 fraction was confirmed by SDS-PAGE and Western blot analysis using an anti-(His)6 antibody (Abcam, #ab18184).

    Techniques: Derivative Assay, Plasmid Preparation, In Vitro, Activity Assay, Mutagenesis, Incubation, TNKS1 Histone Ribosylation Assay, Western Blot, Expressing, In Vivo

    Model for the diphthamide pathway incorporating the proposed novel roles of Dph5, Dph6, and Dph7. (A) Diphthamide pathway showing interaction of Dph5 with unmodified eEF2 and the proposed role of Dph7 in displacement of Dph5 prior to diphthine amidation. (B) Elimination of the trimethylamino group in the absence of the proposed amidase Dph6 suggesting lability of diphthine in its absence.

    Journal: PLoS Genetics

    Article Title: The Amidation Step of Diphthamide Biosynthesis in Yeast Requires DPH6, a Gene Identified through Mining the DPH1-DPH5 Interaction Network

    doi: 10.1371/journal.pgen.1003334

    Figure Lengend Snippet: Model for the diphthamide pathway incorporating the proposed novel roles of Dph5, Dph6, and Dph7. (A) Diphthamide pathway showing interaction of Dph5 with unmodified eEF2 and the proposed role of Dph7 in displacement of Dph5 prior to diphthine amidation. (B) Elimination of the trimethylamino group in the absence of the proposed amidase Dph6 suggesting lability of diphthine in its absence.

    Article Snippet: The identity of purified eEF2 fraction was confirmed by SDS-PAGE and Western blot analysis using an anti-(His)6 antibody (Abcam, #ab18184).

    Techniques: