hela nuclear extract Search Results


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  • 99
    Millipore hela nuclear extract
    Depletion of CTCF in the FXN 5′UTR in FRDA. ( A ) Schematic representation of the relevant portion of the FXN gene showing the CTCF binding site in the 5′UTR, and the regions analyzed by <t>EMSA</t> and ChIP. All numbers are relative to TSS1. The region denoted as “Pro” is the portion of the promoter/5′UTR previously analyzed by other groups and shown not to have heterochromatin formation in FRDA [6] , [8] . ( B ) EMSA with <t>HeLa</t> nuclear extract showing a single complex (arrow) that was competed with excess cold probe, CTCF binding oligonucleotide, but not with a mutant CTCF oligonucleotide, indicating that CTCF binds in the FXN 5′UTR. ( C ) ChIP with anti-CTCF in fibroblasts from non-FRDA controls showing enrichment of CTCF in the FXN 5′UTR in vivo . The CTCF insulator (MYC-N) and a non-binding site (MYC-G) in the MYC gene were used as positive and negative controls for CTCF enrichment in vivo . The enrichment at the FXN 5′UTR is comparable to the MYC locus. The GAPDH fragment used to normalize the ChIP data did not show CTCF enrichment. ( D ) ChIP with anti-CTCF in fibroblasts from two non-FRDA control fibroblasts (CNTR) and two FRDA patients showed significantly less occupancy of CTCF in FRDA. The data are from two independent chromatin preparations, with each experiment done in triplicate. Error bars = s.e.m.; “*” = P
    Hela Nuclear Extract, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam hela nuclear extract
    Differential recruitment of coregulators to ER α upon CE, BAZ, and TSEC treatment. (A) ER α . Proteins associated with ER α after different hormone treatments were identified by MS analyses, and the peptide numbers were listed. (B) After ER α <t>/ERE-DNA</t> pull-down analyses, the levels of SRC-1, SRC-2, SRC-3, BCOR, and ER α in the precipitates were determined by Western blotting analyses. SRC-1 (C), SRC-2 (D), and SRC-3 (E) protein levels in <t>HeLa</t> cells treated with hormone for 24 hours were determined by Western blotting analyses ( n = 3/group). The tubulin level in each group was used as a protein loading control. (F) The ratios of SRCs to tubulin levels in (C)–(E) are shown in a graph. NS, nonspecific, Student’s t test. Values represent the average ± S.E.M. of three independent experiments.
    Hela Nuclear Extract, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzo Biochem hela nuclear extract
    Differential recruitment of coregulators to ER α upon CE, BAZ, and TSEC treatment. (A) ER α . Proteins associated with ER α after different hormone treatments were identified by MS analyses, and the peptide numbers were listed. (B) After ER α <t>/ERE-DNA</t> pull-down analyses, the levels of SRC-1, SRC-2, SRC-3, BCOR, and ER α in the precipitates were determined by Western blotting analyses. SRC-1 (C), SRC-2 (D), and SRC-3 (E) protein levels in <t>HeLa</t> cells treated with hormone for 24 hours were determined by Western blotting analyses ( n = 3/group). The tubulin level in each group was used as a protein loading control. (F) The ratios of SRCs to tubulin levels in (C)–(E) are shown in a graph. NS, nonspecific, Student’s t test. Values represent the average ± S.E.M. of three independent experiments.
    Hela Nuclear Extract, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Promega hela nuclear extract
    Incorporation of cytidine analogues with <t>HeLa</t> nuclear extract. The transcripts were analyzed by denaturing 5% polyacrylamide gel electrophoresis. Above each lane, rNTP added to the extract are recorded. Molar ratios of ATP:( 32 P-)GTP:CTP:UTP:C*TP is 1:0.04:1:1:1 if present ( A ). Two-dimensional cellulose TLC for nearest-neighbor analyses of the transcripts ( B ). In the transcription, the CTP analogues, ho 4 CTP, mo 4 CTP and rPTP, respectively, were added in place of UTP, while sc 4 CTP was added in place of CTP.
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    Promega hela nuclear extracts
    EMSA with <t>HeLa</t> nuclear extracts using <t>-537G</t> and -537A oligonucleotides . Binding activities of [γ- 32 P] ATP-labeled -537G (lane 1–6) and -537A (lane 7–12) oligonucleotides. The assay was performed in the presence (+) or absence (-) of HeLa nuclear extracts. Unlabeled -537G or -537A oligonucleotides were used in competition assays. Each binding reaction contained 5 mg of HeLa nuclear extracts and labeled -537G (lanes 2–6) or -537A (lanes 8–12) oligonucleotides. Excess unlabeled oligonucleotides (10-, 50- and 100-fold) were included in the binding reactions as competitors (Lanes 3–5 and 9–11, respectively). In addition, we added a 100-fold excess of unlabeled -537A and -537G oligonucleotides to compete with -537G (Lane 6) and -537A (Lane 12) oligonucleotides. The binding activity of -537G was unaffected, even in the presence of a 100-fold excess of -537A competitor (lane 6). However, the -537A oligonucleotide could not bind transcription factor (lane 12), and displayed no band in the presence of a 100-fold excess of -537G probe.
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    91
    Upstate Biotechnology Inc hela nuclear extract
    EMSA with <t>HeLa</t> nuclear extracts using <t>-537G</t> and -537A oligonucleotides . Binding activities of [γ- 32 P] ATP-labeled -537G (lane 1–6) and -537A (lane 7–12) oligonucleotides. The assay was performed in the presence (+) or absence (-) of HeLa nuclear extracts. Unlabeled -537G or -537A oligonucleotides were used in competition assays. Each binding reaction contained 5 mg of HeLa nuclear extracts and labeled -537G (lanes 2–6) or -537A (lanes 8–12) oligonucleotides. Excess unlabeled oligonucleotides (10-, 50- and 100-fold) were included in the binding reactions as competitors (Lanes 3–5 and 9–11, respectively). In addition, we added a 100-fold excess of unlabeled -537A and -537G oligonucleotides to compete with -537G (Lane 6) and -537A (Lane 12) oligonucleotides. The binding activity of -537G was unaffected, even in the presence of a 100-fold excess of -537A competitor (lane 6). However, the -537A oligonucleotide could not bind transcription factor (lane 12), and displayed no band in the presence of a 100-fold excess of -537G probe.
    Hela Nuclear Extract, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 91/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Accurate Chemical & Scientific Corporation hela nuclear extract
    Synthetic SR protein binding sites stimulate polyadenylation and CFIm25 crosslinking to the RSV PAS in vitro. A. In vitro polyadenylation assays. Above the panels are representations of the substrates; the larger and smaller boxes indicate regions upstream and downstream of the PAS. The three boxes upstream of the RSV substrate indicate 3x SRSF1 or SRSF7 protein binding sites, or random (RAN) sites. The indicated <t>[α-32P]-labeled</t> substrates were incubated in <t>HeLa</t> nuclear extract for the indicated times and resolved on an 8M urea-6% polyacrylamide gel and imaged with a phosphorimager. The upward smear is polyadenylated RNA. M, size markers. B. Quantitation of data in A as percent polyadenylated RNA (poly(A)/(substrate +poly(A)). Error bars represent standard error of the mean. C. CFIm25 crosslinking to RSV PAS. RNA substrates uniformly labeled with [α-32P] were incubated in HeLa nuclear extract, irradiated with 256nm light (or not, for controls), RNase treated, and samples were incubated with beads pre-bound with anti-NUDIX21 antibody specific for CFIm25 (α-25) or with secondary antibody only (α-mouse). After precipitation and washing, samples were resolved on a 12% SDS- polyacrylamide gel followed by phosphorimaging. XL, the L3 substrate subjected to cross-linking but no immunoprecipitation. Size standards are shown at the right. The arrow points to the CFIm25 band.
    Hela Nuclear Extract, supplied by Accurate Chemical & Scientific Corporation, used in various techniques. Bioz Stars score: 90/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Active Motif of hela nuclear extract
    HDACi screen and antiparasitic and cytotoxicity activities correlation. Screen for <t>anti-HDAC</t> activity (A) . HDAC activity of <t>HeLa</t> nuclear extracts and of recombinant HDAC 1, 3, 6, and 8 proteins was a certained in the presence of 1.0 μM of each of the 8 synthesized compounds, as well as of SAHA. The experiments were carried out in triplicate, and the results are expressed as the percentage of deacetylase activity compared to the untreated control. Inhibition of T. gondii deacetylase activity by SAHA and the compound 363 (B). The total protein extract of T. gondii tachyzoites were prepared as described in the Materials and Methods section. The deacetylase activity was determined after the addition of 0.125 μM of compound 363 or of SAHA. The results are expressed as the percentage of deacetylase activity compared to untreated T. gondii protein extracts. The results are the mean of 4 experiments, carried out in triplicate. Correlation between HDACi and anti T. gondii activity or cytotoxicity potency (C).
    Of Hela Nuclear Extract, supplied by Active Motif, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Active Motif hela nuclear extract
    ( a ) The process of the nuclear extract experiment for 10 μg/well <t>TNF-α–stimulated</t> <t>HeLa</t> Nuclear Extract and 10 μg/well 4-h Serum HeLa Nuclear Extract. The dye-quencher was added to the protein solution at time = 0, and the target DNA was added at time = 197th minute; ( b ) comparison of increased normalized fluorescence values obtained at the 400th and 568th minutes from Figure 4 a, after adding target DNA for 10 μg/well TNF-α–stimulated HeLa Nuclear Extract and 10 μg/well 4-h Serum HeLa Nuclear Extract. The data from Figure 4 a are normalized following Equation (1).
    Hela Nuclear Extract, supplied by Active Motif, used in various techniques. Bioz Stars score: 91/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    BioVision hela cell nuclear extract
    Inhibition of HDACs activity by MPT0E028 and SAHA. (A) Inhibition of HDACs activity in <t>HeLa</t> nuclear extracts. Data were expressed as the mean of at least 3 independent experiments. (B) Inhibition of total <t>HDAC</t> activity by MPT0E028 and SAHA. HCT116 cells were treated with the indicated concentrations of MPT0E028 and SAHA for 24 h, and the nuclear proteins were isolated to determine the inhibition of total HDAC enzyme activity. Data are expressed as the mean±S.E.M. of at least 3 independent experiments.
    Hela Cell Nuclear Extract, supplied by BioVision, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Active Motif hela s3 nuclear extracts
    Inhibition of HDACs activity by MPT0E028 and SAHA. (A) Inhibition of HDACs activity in <t>HeLa</t> nuclear extracts. Data were expressed as the mean of at least 3 independent experiments. (B) Inhibition of total <t>HDAC</t> activity by MPT0E028 and SAHA. HCT116 cells were treated with the indicated concentrations of MPT0E028 and SAHA for 24 h, and the nuclear proteins were isolated to determine the inhibition of total HDAC enzyme activity. Data are expressed as the mean±S.E.M. of at least 3 independent experiments.
    Hela S3 Nuclear Extracts, supplied by Active Motif, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega hela cell nuclear extract
    CTD phosphorylation using immunopurified proteins with antibodies to TFIIH subunits. (A) Antibodies to TFIIH subunits ERCC3 (p89) (αERCC3) and p62 (αp62) or to TBP (αTBP) were used for immunoprecipitation from the <t>HeLa</t> cell nuclear extract. These <t>immunoprecipitates</t> were then used in kinase assays to phosphorylate GST-CTD, resulting in 32 P-labeled GST-CTD o and GST-CTD a as indicated. Molecular mass markers (in kilodaltons) are indicated to the left. (B) Antibodies to TFIIH subunit p62, (αp62), cyclin H, (αcyclinH), or MO15 (cdk7) (αMO15) were used to immunopurify proteins from untreated (−) or cdc2-treated (+) HeLa cell nuclear extract. Kinase assays were performed with GST-CTD as a substrate.
    Hela Cell Nuclear Extract, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 94 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology hela cell nuclear extracts
    Corepressor complexes implicated in TR repression contain no detectable HMT activity. (A) The corepressor complexes Sin3, <t>SMRT,</t> N-CoR, and Mi-2/NURD were individually immunoprecipitated from <t>HeLa</t> nuclear extracts with complex-specific antibodies as indicated and tested for HMT activity with core histones as substrates. Note that no detectable HMT activity was found in each immunoprecipitated complex, although HeLa nuclear extract (lane 1) contained abundant HMT activity toward H3 and less for H4. Also shown is Coomassie staining of core histones. In contrast, all immunoprecipitated corepressor complexes exhibited HDAC activity, as revealed by an in vitro deacetylation assay (B). Lane 1, immunoprecipitation with rabbit immunoglobulin G as a control for the deacetylation assay.
    Hela Cell Nuclear Extracts, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Accurate Chemical & Scientific Corporation hela cell nuclear extract
    Corepressor complexes implicated in TR repression contain no detectable HMT activity. (A) The corepressor complexes Sin3, <t>SMRT,</t> N-CoR, and Mi-2/NURD were individually immunoprecipitated from <t>HeLa</t> nuclear extracts with complex-specific antibodies as indicated and tested for HMT activity with core histones as substrates. Note that no detectable HMT activity was found in each immunoprecipitated complex, although HeLa nuclear extract (lane 1) contained abundant HMT activity toward H3 and less for H4. Also shown is Coomassie staining of core histones. In contrast, all immunoprecipitated corepressor complexes exhibited HDAC activity, as revealed by an in vitro deacetylation assay (B). Lane 1, immunoprecipitation with rabbit immunoglobulin G as a control for the deacetylation assay.
    Hela Cell Nuclear Extract, supplied by Accurate Chemical & Scientific Corporation, used in various techniques. Bioz Stars score: 89/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology hela cell nuclear extract
    Corepressor complexes implicated in TR repression contain no detectable HMT activity. (A) The corepressor complexes Sin3, <t>SMRT,</t> N-CoR, and Mi-2/NURD were individually immunoprecipitated from <t>HeLa</t> nuclear extracts with complex-specific antibodies as indicated and tested for HMT activity with core histones as substrates. Note that no detectable HMT activity was found in each immunoprecipitated complex, although HeLa nuclear extract (lane 1) contained abundant HMT activity toward H3 and less for H4. Also shown is Coomassie staining of core histones. In contrast, all immunoprecipitated corepressor complexes exhibited HDAC activity, as revealed by an in vitro deacetylation assay (B). Lane 1, immunoprecipitation with rabbit immunoglobulin G as a control for the deacetylation assay.
    Hela Cell Nuclear Extract, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene hela cell nuclear extracts
    Corepressor complexes implicated in TR repression contain no detectable HMT activity. (A) The corepressor complexes Sin3, <t>SMRT,</t> N-CoR, and Mi-2/NURD were individually immunoprecipitated from <t>HeLa</t> nuclear extracts with complex-specific antibodies as indicated and tested for HMT activity with core histones as substrates. Note that no detectable HMT activity was found in each immunoprecipitated complex, although HeLa nuclear extract (lane 1) contained abundant HMT activity toward H3 and less for H4. Also shown is Coomassie staining of core histones. In contrast, all immunoprecipitated corepressor complexes exhibited HDAC activity, as revealed by an in vitro deacetylation assay (B). Lane 1, immunoprecipitation with rabbit immunoglobulin G as a control for the deacetylation assay.
    Hela Cell Nuclear Extracts, supplied by Stratagene, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology hela nuclear extract
    PKR is in splicing complexes and required for splicing and spliceosome formation. (A) β-globin pre-mRNA template was incubated in <t>HeLa</t> cell nuclear extract for 30 min before <t>immunoprecipitation</t> with NGS, αSC35 or αPKR. Western blots of PKR (top) and SC35 (bottom) are shown for soluble (Sup) and precipitated fractions (Pellet). (B) Anti-PKR Abs inhibit spliceosome formation. Labeled β-globin pre-mRNA template was incubated in HeLa cell nuclear extract for the times shown. Where indicated, αPKR cat or αPKR rbd was added 20 min before incubation. Ribonucleoprotein splicing complexes A, B, C and H were resolved by native PAGE. Left lane shows input unspliced RNA. (C - D) Recombinant PKR (rPKR) reconstitutes β-globin pre-mRNA splicing upon PKR depletion. (C) After adding NaCl to 500 mM, nuclear extract was incubated at 4 °C with αIgG or αPKR Ab for 18 h; the immunoprecipitate was removed using protein A/G Sepharose beads. The depleted extract was centrifuged through Sephadex G-25 before in vitro splicing was performed for 2 h, with rPKR or storage buffer (buffer) added as shown. (D) Plot of mRNA/pre-mRNA ratio in C ; αIgG, grey bars; αPKR, black bars. (E) In parallel, splicing of MINX RNA was assayed as in C . U, input unspliced RNA. (F) Reconstitution of spliceosomes on β-globin pre-mRNA template with rPKR. Formation of spliceosomes was assayed as in B , using extract depleted of PKR as in C . Where indicated, 480 ng of rPKR was added back. Three lanes on right show MINX RNA, assayed in parallel.
    Hela Nuclear Extract, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Upstate Biotechnology Inc hela nuclear extracts
    PKR is in splicing complexes and required for splicing and spliceosome formation. (A) β-globin pre-mRNA template was incubated in <t>HeLa</t> cell nuclear extract for 30 min before <t>immunoprecipitation</t> with NGS, αSC35 or αPKR. Western blots of PKR (top) and SC35 (bottom) are shown for soluble (Sup) and precipitated fractions (Pellet). (B) Anti-PKR Abs inhibit spliceosome formation. Labeled β-globin pre-mRNA template was incubated in HeLa cell nuclear extract for the times shown. Where indicated, αPKR cat or αPKR rbd was added 20 min before incubation. Ribonucleoprotein splicing complexes A, B, C and H were resolved by native PAGE. Left lane shows input unspliced RNA. (C - D) Recombinant PKR (rPKR) reconstitutes β-globin pre-mRNA splicing upon PKR depletion. (C) After adding NaCl to 500 mM, nuclear extract was incubated at 4 °C with αIgG or αPKR Ab for 18 h; the immunoprecipitate was removed using protein A/G Sepharose beads. The depleted extract was centrifuged through Sephadex G-25 before in vitro splicing was performed for 2 h, with rPKR or storage buffer (buffer) added as shown. (D) Plot of mRNA/pre-mRNA ratio in C ; αIgG, grey bars; αPKR, black bars. (E) In parallel, splicing of MINX RNA was assayed as in C . U, input unspliced RNA. (F) Reconstitution of spliceosomes on β-globin pre-mRNA template with rPKR. Formation of spliceosomes was assayed as in B , using extract depleted of PKR as in C . Where indicated, 480 ng of rPKR was added back. Three lanes on right show MINX RNA, assayed in parallel.
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    AnaSpec hela nuclear extract
    PKR is in splicing complexes and required for splicing and spliceosome formation. (A) β-globin pre-mRNA template was incubated in <t>HeLa</t> cell nuclear extract for 30 min before <t>immunoprecipitation</t> with NGS, αSC35 or αPKR. Western blots of PKR (top) and SC35 (bottom) are shown for soluble (Sup) and precipitated fractions (Pellet). (B) Anti-PKR Abs inhibit spliceosome formation. Labeled β-globin pre-mRNA template was incubated in HeLa cell nuclear extract for the times shown. Where indicated, αPKR cat or αPKR rbd was added 20 min before incubation. Ribonucleoprotein splicing complexes A, B, C and H were resolved by native PAGE. Left lane shows input unspliced RNA. (C - D) Recombinant PKR (rPKR) reconstitutes β-globin pre-mRNA splicing upon PKR depletion. (C) After adding NaCl to 500 mM, nuclear extract was incubated at 4 °C with αIgG or αPKR Ab for 18 h; the immunoprecipitate was removed using protein A/G Sepharose beads. The depleted extract was centrifuged through Sephadex G-25 before in vitro splicing was performed for 2 h, with rPKR or storage buffer (buffer) added as shown. (D) Plot of mRNA/pre-mRNA ratio in C ; αIgG, grey bars; αPKR, black bars. (E) In parallel, splicing of MINX RNA was assayed as in C . U, input unspliced RNA. (F) Reconstitution of spliceosomes on β-globin pre-mRNA template with rPKR. Formation of spliceosomes was assayed as in B , using extract depleted of PKR as in C . Where indicated, 480 ng of rPKR was added back. Three lanes on right show MINX RNA, assayed in parallel.
    Hela Nuclear Extract, supplied by AnaSpec, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Depletion of CTCF in the FXN 5′UTR in FRDA. ( A ) Schematic representation of the relevant portion of the FXN gene showing the CTCF binding site in the 5′UTR, and the regions analyzed by EMSA and ChIP. All numbers are relative to TSS1. The region denoted as “Pro” is the portion of the promoter/5′UTR previously analyzed by other groups and shown not to have heterochromatin formation in FRDA [6] , [8] . ( B ) EMSA with HeLa nuclear extract showing a single complex (arrow) that was competed with excess cold probe, CTCF binding oligonucleotide, but not with a mutant CTCF oligonucleotide, indicating that CTCF binds in the FXN 5′UTR. ( C ) ChIP with anti-CTCF in fibroblasts from non-FRDA controls showing enrichment of CTCF in the FXN 5′UTR in vivo . The CTCF insulator (MYC-N) and a non-binding site (MYC-G) in the MYC gene were used as positive and negative controls for CTCF enrichment in vivo . The enrichment at the FXN 5′UTR is comparable to the MYC locus. The GAPDH fragment used to normalize the ChIP data did not show CTCF enrichment. ( D ) ChIP with anti-CTCF in fibroblasts from two non-FRDA control fibroblasts (CNTR) and two FRDA patients showed significantly less occupancy of CTCF in FRDA. The data are from two independent chromatin preparations, with each experiment done in triplicate. Error bars = s.e.m.; “*” = P

    Journal: PLoS ONE

    Article Title: Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription

    doi: 10.1371/journal.pone.0007914

    Figure Lengend Snippet: Depletion of CTCF in the FXN 5′UTR in FRDA. ( A ) Schematic representation of the relevant portion of the FXN gene showing the CTCF binding site in the 5′UTR, and the regions analyzed by EMSA and ChIP. All numbers are relative to TSS1. The region denoted as “Pro” is the portion of the promoter/5′UTR previously analyzed by other groups and shown not to have heterochromatin formation in FRDA [6] , [8] . ( B ) EMSA with HeLa nuclear extract showing a single complex (arrow) that was competed with excess cold probe, CTCF binding oligonucleotide, but not with a mutant CTCF oligonucleotide, indicating that CTCF binds in the FXN 5′UTR. ( C ) ChIP with anti-CTCF in fibroblasts from non-FRDA controls showing enrichment of CTCF in the FXN 5′UTR in vivo . The CTCF insulator (MYC-N) and a non-binding site (MYC-G) in the MYC gene were used as positive and negative controls for CTCF enrichment in vivo . The enrichment at the FXN 5′UTR is comparable to the MYC locus. The GAPDH fragment used to normalize the ChIP data did not show CTCF enrichment. ( D ) ChIP with anti-CTCF in fibroblasts from two non-FRDA control fibroblasts (CNTR) and two FRDA patients showed significantly less occupancy of CTCF in FRDA. The data are from two independent chromatin preparations, with each experiment done in triplicate. Error bars = s.e.m.; “*” = P

    Article Snippet: EMSA performed with HeLa nuclear extract and an in vitro methylated 5′UTR probe (methylation status of which was confirmed by bisulfite sequencing) showed a similar major complex as with the unmethylated probe.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Mutagenesis, In Vivo

    Differential recruitment of coregulators to ER α upon CE, BAZ, and TSEC treatment. (A) ER α . Proteins associated with ER α after different hormone treatments were identified by MS analyses, and the peptide numbers were listed. (B) After ER α /ERE-DNA pull-down analyses, the levels of SRC-1, SRC-2, SRC-3, BCOR, and ER α in the precipitates were determined by Western blotting analyses. SRC-1 (C), SRC-2 (D), and SRC-3 (E) protein levels in HeLa cells treated with hormone for 24 hours were determined by Western blotting analyses ( n = 3/group). The tubulin level in each group was used as a protein loading control. (F) The ratios of SRCs to tubulin levels in (C)–(E) are shown in a graph. NS, nonspecific, Student’s t test. Values represent the average ± S.E.M. of three independent experiments.

    Journal: Molecular Pharmacology

    Article Title: The Dual Estrogen Receptor α

    doi: 10.1124/mol.115.100925

    Figure Lengend Snippet: Differential recruitment of coregulators to ER α upon CE, BAZ, and TSEC treatment. (A) ER α . Proteins associated with ER α after different hormone treatments were identified by MS analyses, and the peptide numbers were listed. (B) After ER α /ERE-DNA pull-down analyses, the levels of SRC-1, SRC-2, SRC-3, BCOR, and ER α in the precipitates were determined by Western blotting analyses. SRC-1 (C), SRC-2 (D), and SRC-3 (E) protein levels in HeLa cells treated with hormone for 24 hours were determined by Western blotting analyses ( n = 3/group). The tubulin level in each group was used as a protein loading control. (F) The ratios of SRCs to tubulin levels in (C)–(E) are shown in a graph. NS, nonspecific, Student’s t test. Values represent the average ± S.E.M. of three independent experiments.

    Article Snippet: In brief, 4 µ g of ERE DNA, 1 mg of HeLa nuclear extract, 0.5 µ g of recombinant human ER α protein (Abcam), and 60 μ l of Dynabeads M-280 Streptavidin were mixed with NETN (0.5% N onyl Phenoxypolyethoxylethanol (NP)-40, 1 mM E DTA, 50 mM T ris-HCl (PH7.5) and 150 mM N aCl) buffer and then incubated with vehicle, 10 nM CE, 100 nM BAZ, and 10 nM CE + 100 nM BAZ (TSEC) at 4°C for 1.5 hours with constant shaking.

    Techniques: Mass Spectrometry, Western Blot

    ER α interacts with FBXO45 upon TSEC treatment. (A and B) HeLa cells in a six-well plate were transfected with 100 ng of ER α expression vector. After 48 hours, cells were treated with vehicle, CE (10 nM), BAZ (100 nM), and TSEC (10 nM CE plus 100 nM BAZ) for 24 hours in the absence (A) or presence (B) of MG132 (10 µ M). Subsequently, the ER α and tubulin protein levels in HeLa cells were determined by Western blotting analyses ( n = 2/group). The ratios of ER α to tubulin in HeLa cells are shown graphically ( n = 4/group). (C) Peptide numbers of the ubiquitin ligases that were specifically associated with TSEC-bound ER α after ER α /ERE-DNA pull-down analyses. (D) ER α /ERE-DNA pull-down analysis was performed in the presence of hormone, and the FBXO45 levels in the precipitant were determined by Western blotting analyses. (E) HeLa cells were transiently transfected with expression vectors for ER α and/or Flag-tagged FBXO45. After 48 hours, anti-Flag antibodies were used to immunoprecipitate Flag-tagged FBXO45 from each cell lysate in the absence or presence of MG132 (10 µ M). The levels of ER α and FBXO45 in the immunoprecipitates were determined by Western blotting analyses. (F) HeLa cells were transiently transfected with expression vectors for ER α and Flag-FBXO45. After 48 hours, cells were treated with vehicle, CE (10 nM), BAZ (100 nM), and TSEC (10 nM CE plus 100 nM BAZ) for 6 hours. Using anti-Flag antibodies, Flag-FBXO45 was immunoprecipitated from each group, and the ER α levels in the immunoprecipitates were determined by Western blotting analyses. *** P

    Journal: Molecular Pharmacology

    Article Title: The Dual Estrogen Receptor α

    doi: 10.1124/mol.115.100925

    Figure Lengend Snippet: ER α interacts with FBXO45 upon TSEC treatment. (A and B) HeLa cells in a six-well plate were transfected with 100 ng of ER α expression vector. After 48 hours, cells were treated with vehicle, CE (10 nM), BAZ (100 nM), and TSEC (10 nM CE plus 100 nM BAZ) for 24 hours in the absence (A) or presence (B) of MG132 (10 µ M). Subsequently, the ER α and tubulin protein levels in HeLa cells were determined by Western blotting analyses ( n = 2/group). The ratios of ER α to tubulin in HeLa cells are shown graphically ( n = 4/group). (C) Peptide numbers of the ubiquitin ligases that were specifically associated with TSEC-bound ER α after ER α /ERE-DNA pull-down analyses. (D) ER α /ERE-DNA pull-down analysis was performed in the presence of hormone, and the FBXO45 levels in the precipitant were determined by Western blotting analyses. (E) HeLa cells were transiently transfected with expression vectors for ER α and/or Flag-tagged FBXO45. After 48 hours, anti-Flag antibodies were used to immunoprecipitate Flag-tagged FBXO45 from each cell lysate in the absence or presence of MG132 (10 µ M). The levels of ER α and FBXO45 in the immunoprecipitates were determined by Western blotting analyses. (F) HeLa cells were transiently transfected with expression vectors for ER α and Flag-FBXO45. After 48 hours, cells were treated with vehicle, CE (10 nM), BAZ (100 nM), and TSEC (10 nM CE plus 100 nM BAZ) for 6 hours. Using anti-Flag antibodies, Flag-FBXO45 was immunoprecipitated from each group, and the ER α levels in the immunoprecipitates were determined by Western blotting analyses. *** P

    Article Snippet: In brief, 4 µ g of ERE DNA, 1 mg of HeLa nuclear extract, 0.5 µ g of recombinant human ER α protein (Abcam), and 60 μ l of Dynabeads M-280 Streptavidin were mixed with NETN (0.5% N onyl Phenoxypolyethoxylethanol (NP)-40, 1 mM E DTA, 50 mM T ris-HCl (PH7.5) and 150 mM N aCl) buffer and then incubated with vehicle, 10 nM CE, 100 nM BAZ, and 10 nM CE + 100 nM BAZ (TSEC) at 4°C for 1.5 hours with constant shaking.

    Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation

    Incorporation of cytidine analogues with HeLa nuclear extract. The transcripts were analyzed by denaturing 5% polyacrylamide gel electrophoresis. Above each lane, rNTP added to the extract are recorded. Molar ratios of ATP:( 32 P-)GTP:CTP:UTP:C*TP is 1:0.04:1:1:1 if present ( A ). Two-dimensional cellulose TLC for nearest-neighbor analyses of the transcripts ( B ). In the transcription, the CTP analogues, ho 4 CTP, mo 4 CTP and rPTP, respectively, were added in place of UTP, while sc 4 CTP was added in place of CTP.

    Journal: Nucleic Acids Research

    Article Title: Template properties of mutagenic cytosine analogues in reverse transcription

    doi: 10.1093/nar/gkl761

    Figure Lengend Snippet: Incorporation of cytidine analogues with HeLa nuclear extract. The transcripts were analyzed by denaturing 5% polyacrylamide gel electrophoresis. Above each lane, rNTP added to the extract are recorded. Molar ratios of ATP:( 32 P-)GTP:CTP:UTP:C*TP is 1:0.04:1:1:1 if present ( A ). Two-dimensional cellulose TLC for nearest-neighbor analyses of the transcripts ( B ). In the transcription, the CTP analogues, ho 4 CTP, mo 4 CTP and rPTP, respectively, were added in place of UTP, while sc 4 CTP was added in place of CTP.

    Article Snippet: A standard reaction mixture (25 μl) contained 20 mM HEPES (pH 7.9), 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 3 mM MgCl2 , 0.4 mM ATP, CTP and UTP, 0.016 mM GTP, 450 ng DNA template, 40 U RNase inhibitor, 10 μCi [α-32 P]GTP (3000 Ci/mmol) and 8 U HeLa nuclear extract.

    Techniques: Polyacrylamide Gel Electrophoresis, Thin Layer Chromatography

    EMSA with HeLa nuclear extracts using -537G and -537A oligonucleotides . Binding activities of [γ- 32 P] ATP-labeled -537G (lane 1–6) and -537A (lane 7–12) oligonucleotides. The assay was performed in the presence (+) or absence (-) of HeLa nuclear extracts. Unlabeled -537G or -537A oligonucleotides were used in competition assays. Each binding reaction contained 5 mg of HeLa nuclear extracts and labeled -537G (lanes 2–6) or -537A (lanes 8–12) oligonucleotides. Excess unlabeled oligonucleotides (10-, 50- and 100-fold) were included in the binding reactions as competitors (Lanes 3–5 and 9–11, respectively). In addition, we added a 100-fold excess of unlabeled -537A and -537G oligonucleotides to compete with -537G (Lane 6) and -537A (Lane 12) oligonucleotides. The binding activity of -537G was unaffected, even in the presence of a 100-fold excess of -537A competitor (lane 6). However, the -537A oligonucleotide could not bind transcription factor (lane 12), and displayed no band in the presence of a 100-fold excess of -537G probe.

    Journal: BMC Cancer

    Article Title: GSTT2 promoter polymorphisms and colorectal cancer risk

    doi: 10.1186/1471-2407-7-16

    Figure Lengend Snippet: EMSA with HeLa nuclear extracts using -537G and -537A oligonucleotides . Binding activities of [γ- 32 P] ATP-labeled -537G (lane 1–6) and -537A (lane 7–12) oligonucleotides. The assay was performed in the presence (+) or absence (-) of HeLa nuclear extracts. Unlabeled -537G or -537A oligonucleotides were used in competition assays. Each binding reaction contained 5 mg of HeLa nuclear extracts and labeled -537G (lanes 2–6) or -537A (lanes 8–12) oligonucleotides. Excess unlabeled oligonucleotides (10-, 50- and 100-fold) were included in the binding reactions as competitors (Lanes 3–5 and 9–11, respectively). In addition, we added a 100-fold excess of unlabeled -537A and -537G oligonucleotides to compete with -537G (Lane 6) and -537A (Lane 12) oligonucleotides. The binding activity of -537G was unaffected, even in the presence of a 100-fold excess of -537A competitor (lane 6). However, the -537A oligonucleotide could not bind transcription factor (lane 12), and displayed no band in the presence of a 100-fold excess of -537G probe.

    Article Snippet: EMSA was performed to investigate the binding between the -537G and A alleles in HeLa nuclear extracts.

    Techniques: Binding Assay, Labeling, Activity Assay

    C. albicans TBP function in vitro. (A) SDS-PAGE analysis of 35 S-TBP generated by in vitro transcription and translation of the C. albicans TBP1 ORF (Materials and Methods). Lane 1, control reaction mixture containing no TBP1 cDNA; lane 2, lysate containing TBP1 cDNA showing the 35 S-TBP polypeptide with an M r of 27,000. (B) Gel-shift assays comparing in vitro-synthesized C. albicans TBP (CaTBP) with HeLa cell extracts containing TBP. These were incubated with synthetic double-stranded oligonucleotides carrying a HeLa TATA box, the C. albicans HYR1 TATA box, or a control oligonucleotide lacking TATA (STRE). Competitor was a 10-fold excess of unlabelled HeLa or HYR1 TATA oligonucleotide. Closed arrowhead, complex formation; open arrowheads, uncomplexed oligonucleotides. (C) PAGE analysis of 32 P-labelled in vitro transcription products. Transcription from the cytomegalovirus immediate-early promoter was performed with HeLa cell nuclear extracts with (+) or without (−) prior heat treatment to inactivate endogenous TBP. CaTBP, in vitro-synthesized C. albicans TBP; M, pBR322- Msp I size markers.

    Journal: Journal of Bacteriology

    Article Title: The TATA-Binding Protein (TBP) from the Human Fungal Pathogen Candida albicans Can Complement Defects in Human and Yeast TBPs

    doi:

    Figure Lengend Snippet: C. albicans TBP function in vitro. (A) SDS-PAGE analysis of 35 S-TBP generated by in vitro transcription and translation of the C. albicans TBP1 ORF (Materials and Methods). Lane 1, control reaction mixture containing no TBP1 cDNA; lane 2, lysate containing TBP1 cDNA showing the 35 S-TBP polypeptide with an M r of 27,000. (B) Gel-shift assays comparing in vitro-synthesized C. albicans TBP (CaTBP) with HeLa cell extracts containing TBP. These were incubated with synthetic double-stranded oligonucleotides carrying a HeLa TATA box, the C. albicans HYR1 TATA box, or a control oligonucleotide lacking TATA (STRE). Competitor was a 10-fold excess of unlabelled HeLa or HYR1 TATA oligonucleotide. Closed arrowhead, complex formation; open arrowheads, uncomplexed oligonucleotides. (C) PAGE analysis of 32 P-labelled in vitro transcription products. Transcription from the cytomegalovirus immediate-early promoter was performed with HeLa cell nuclear extracts with (+) or without (−) prior heat treatment to inactivate endogenous TBP. CaTBP, in vitro-synthesized C. albicans TBP; M, pBR322- Msp I size markers.

    Article Snippet: Gel mobility shift assays were performed ( , , , ) with coupled transcription-translation reaction mixtures containing C. albicans TBP (see above) or HeLa cell nuclear extracts (Promega).

    Techniques: In Vitro, SDS Page, Generated, Electrophoretic Mobility Shift Assay, Synthesized, Incubation, Polyacrylamide Gel Electrophoresis

    Synthetic SR protein binding sites stimulate polyadenylation and CFIm25 crosslinking to the RSV PAS in vitro. A. In vitro polyadenylation assays. Above the panels are representations of the substrates; the larger and smaller boxes indicate regions upstream and downstream of the PAS. The three boxes upstream of the RSV substrate indicate 3x SRSF1 or SRSF7 protein binding sites, or random (RAN) sites. The indicated [α-32P]-labeled substrates were incubated in HeLa nuclear extract for the indicated times and resolved on an 8M urea-6% polyacrylamide gel and imaged with a phosphorimager. The upward smear is polyadenylated RNA. M, size markers. B. Quantitation of data in A as percent polyadenylated RNA (poly(A)/(substrate +poly(A)). Error bars represent standard error of the mean. C. CFIm25 crosslinking to RSV PAS. RNA substrates uniformly labeled with [α-32P] were incubated in HeLa nuclear extract, irradiated with 256nm light (or not, for controls), RNase treated, and samples were incubated with beads pre-bound with anti-NUDIX21 antibody specific for CFIm25 (α-25) or with secondary antibody only (α-mouse). After precipitation and washing, samples were resolved on a 12% SDS- polyacrylamide gel followed by phosphorimaging. XL, the L3 substrate subjected to cross-linking but no immunoprecipitation. Size standards are shown at the right. The arrow points to the CFIm25 band.

    Journal: Virology

    Article Title: Evidence That a Threshold of Serine/Arginine-Rich (SR) Proteins Recruits CFIm to Promote Rous Sarcoma Virus mRNA 3′ End Formation

    doi: 10.1016/j.virol.2016.08.021

    Figure Lengend Snippet: Synthetic SR protein binding sites stimulate polyadenylation and CFIm25 crosslinking to the RSV PAS in vitro. A. In vitro polyadenylation assays. Above the panels are representations of the substrates; the larger and smaller boxes indicate regions upstream and downstream of the PAS. The three boxes upstream of the RSV substrate indicate 3x SRSF1 or SRSF7 protein binding sites, or random (RAN) sites. The indicated [α-32P]-labeled substrates were incubated in HeLa nuclear extract for the indicated times and resolved on an 8M urea-6% polyacrylamide gel and imaged with a phosphorimager. The upward smear is polyadenylated RNA. M, size markers. B. Quantitation of data in A as percent polyadenylated RNA (poly(A)/(substrate +poly(A)). Error bars represent standard error of the mean. C. CFIm25 crosslinking to RSV PAS. RNA substrates uniformly labeled with [α-32P] were incubated in HeLa nuclear extract, irradiated with 256nm light (or not, for controls), RNase treated, and samples were incubated with beads pre-bound with anti-NUDIX21 antibody specific for CFIm25 (α-25) or with secondary antibody only (α-mouse). After precipitation and washing, samples were resolved on a 12% SDS- polyacrylamide gel followed by phosphorimaging. XL, the L3 substrate subjected to cross-linking but no immunoprecipitation. Size standards are shown at the right. The arrow points to the CFIm25 band.

    Article Snippet: UV-cross-linking assays were conducted using 100,000 cpm of [α-32P]UTP-labeled RNA substrate and 15 μg of HeLa nuclear extract (Accurate Chemical & Scientific Corp.) under splicing conditions as previously described , but in the absence of creatine phosphate and ATP.

    Techniques: Protein Binding, In Vitro, Labeling, Incubation, Quantitation Assay, Irradiation, Immunoprecipitation

    HDACi screen and antiparasitic and cytotoxicity activities correlation. Screen for anti-HDAC activity (A) . HDAC activity of HeLa nuclear extracts and of recombinant HDAC 1, 3, 6, and 8 proteins was a certained in the presence of 1.0 μM of each of the 8 synthesized compounds, as well as of SAHA. The experiments were carried out in triplicate, and the results are expressed as the percentage of deacetylase activity compared to the untreated control. Inhibition of T. gondii deacetylase activity by SAHA and the compound 363 (B). The total protein extract of T. gondii tachyzoites were prepared as described in the Materials and Methods section. The deacetylase activity was determined after the addition of 0.125 μM of compound 363 or of SAHA. The results are expressed as the percentage of deacetylase activity compared to untreated T. gondii protein extracts. The results are the mean of 4 experiments, carried out in triplicate. Correlation between HDACi and anti T. gondii activity or cytotoxicity potency (C).

    Journal: International Journal for Parasitology: Drugs and Drug Resistance

    Article Title: Synthesis of aminophenylhydroxamate and aminobenzylhydroxamate derivatives and in vitro screening for antiparasitic and histone deacetylase inhibitory activity

    doi: 10.1016/j.ijpddr.2018.01.002

    Figure Lengend Snippet: HDACi screen and antiparasitic and cytotoxicity activities correlation. Screen for anti-HDAC activity (A) . HDAC activity of HeLa nuclear extracts and of recombinant HDAC 1, 3, 6, and 8 proteins was a certained in the presence of 1.0 μM of each of the 8 synthesized compounds, as well as of SAHA. The experiments were carried out in triplicate, and the results are expressed as the percentage of deacetylase activity compared to the untreated control. Inhibition of T. gondii deacetylase activity by SAHA and the compound 363 (B). The total protein extract of T. gondii tachyzoites were prepared as described in the Materials and Methods section. The deacetylase activity was determined after the addition of 0.125 μM of compound 363 or of SAHA. The results are expressed as the percentage of deacetylase activity compared to untreated T. gondii protein extracts. The results are the mean of 4 experiments, carried out in triplicate. Correlation between HDACi and anti T. gondii activity or cytotoxicity potency (C).

    Article Snippet: Briefly, 30 μL of HeLa nuclear extract (Active Motif, Belgium) or various HDAC recombinant proteins (Active Motif, Belgium) was mixed with 5 μL of a 10 × compound and 10 μL of assay buffer.

    Techniques: Activity Assay, Recombinant, Synthesized, Histone Deacetylase Assay, Inhibition

    ( a ) The process of the nuclear extract experiment for 10 μg/well TNF-α–stimulated HeLa Nuclear Extract and 10 μg/well 4-h Serum HeLa Nuclear Extract. The dye-quencher was added to the protein solution at time = 0, and the target DNA was added at time = 197th minute; ( b ) comparison of increased normalized fluorescence values obtained at the 400th and 568th minutes from Figure 4 a, after adding target DNA for 10 μg/well TNF-α–stimulated HeLa Nuclear Extract and 10 μg/well 4-h Serum HeLa Nuclear Extract. The data from Figure 4 a are normalized following Equation (1).

    Journal: Biosensors

    Article Title: Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B

    doi: 10.3390/bios8040126

    Figure Lengend Snippet: ( a ) The process of the nuclear extract experiment for 10 μg/well TNF-α–stimulated HeLa Nuclear Extract and 10 μg/well 4-h Serum HeLa Nuclear Extract. The dye-quencher was added to the protein solution at time = 0, and the target DNA was added at time = 197th minute; ( b ) comparison of increased normalized fluorescence values obtained at the 400th and 568th minutes from Figure 4 a, after adding target DNA for 10 μg/well TNF-α–stimulated HeLa Nuclear Extract and 10 μg/well 4-h Serum HeLa Nuclear Extract. The data from Figure 4 a are normalized following Equation (1).

    Article Snippet: Nuclear Extracts Experiment Two kinds of nuclear extracts, HeLa nuclear extract (4-h serum response, with positive transcription factors: c-Fos, Sp1, and SRF) and HeLa nuclear extract (TNF-α stimulated, with positive transcription factor: NF-κB), can be bought from Active Motif Company.

    Techniques: Fluorescence

    Inhibition of HDACs activity by MPT0E028 and SAHA. (A) Inhibition of HDACs activity in HeLa nuclear extracts. Data were expressed as the mean of at least 3 independent experiments. (B) Inhibition of total HDAC activity by MPT0E028 and SAHA. HCT116 cells were treated with the indicated concentrations of MPT0E028 and SAHA for 24 h, and the nuclear proteins were isolated to determine the inhibition of total HDAC enzyme activity. Data are expressed as the mean±S.E.M. of at least 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Anticancer Activity of MPT0E028, a Novel Potent Histone Deacetylase Inhibitor, in Human Colorectal Cancer HCT116 Cells In Vitro and In Vivo

    doi: 10.1371/journal.pone.0043645

    Figure Lengend Snippet: Inhibition of HDACs activity by MPT0E028 and SAHA. (A) Inhibition of HDACs activity in HeLa nuclear extracts. Data were expressed as the mean of at least 3 independent experiments. (B) Inhibition of total HDAC activity by MPT0E028 and SAHA. HCT116 cells were treated with the indicated concentrations of MPT0E028 and SAHA for 24 h, and the nuclear proteins were isolated to determine the inhibition of total HDAC enzyme activity. Data are expressed as the mean±S.E.M. of at least 3 independent experiments.

    Article Snippet: HeLa Nuclear Extract HDAC Activity Assay The HeLa nuclear extract HDAC activity was measured by using the HDAC Fluorescent Activity Assay Kit (BioVision, CA, USA) according to manufacturer’s instructions .

    Techniques: Inhibition, Activity Assay, Isolation

    CTD phosphorylation using immunopurified proteins with antibodies to TFIIH subunits. (A) Antibodies to TFIIH subunits ERCC3 (p89) (αERCC3) and p62 (αp62) or to TBP (αTBP) were used for immunoprecipitation from the HeLa cell nuclear extract. These immunoprecipitates were then used in kinase assays to phosphorylate GST-CTD, resulting in 32 P-labeled GST-CTD o and GST-CTD a as indicated. Molecular mass markers (in kilodaltons) are indicated to the left. (B) Antibodies to TFIIH subunit p62, (αp62), cyclin H, (αcyclinH), or MO15 (cdk7) (αMO15) were used to immunopurify proteins from untreated (−) or cdc2-treated (+) HeLa cell nuclear extract. Kinase assays were performed with GST-CTD as a substrate.

    Journal: Molecular and Cellular Biology

    Article Title: Repression of TFIIH Transcriptional Activity and TFIIH-Associated cdk7 Kinase Activity at Mitosis

    doi:

    Figure Lengend Snippet: CTD phosphorylation using immunopurified proteins with antibodies to TFIIH subunits. (A) Antibodies to TFIIH subunits ERCC3 (p89) (αERCC3) and p62 (αp62) or to TBP (αTBP) were used for immunoprecipitation from the HeLa cell nuclear extract. These immunoprecipitates were then used in kinase assays to phosphorylate GST-CTD, resulting in 32 P-labeled GST-CTD o and GST-CTD a as indicated. Molecular mass markers (in kilodaltons) are indicated to the left. (B) Antibodies to TFIIH subunit p62, (αp62), cyclin H, (αcyclinH), or MO15 (cdk7) (αMO15) were used to immunopurify proteins from untreated (−) or cdc2-treated (+) HeLa cell nuclear extract. Kinase assays were performed with GST-CTD as a substrate.

    Article Snippet: As a control, immunoprecipitates from the HeLa cell nuclear extract, prepared with an antibody to TBP, showed only very low levels of CTD phosphorylation.

    Techniques: Immunoprecipitation, Labeling

    cdc2 inhibition of TFIIH kinase activity is reversed by the cyclin-dependent kinase inhibitor p21. (A) Affinity-purified cdc2/GST-cyclin B was incubated with the indicated concentrations of p21, and these mixtures were then used in kinase reactions with histone H1 as a substrate. The position of 32 P-labeled histone H1 is indicated. The positions of molecular mass markers (in kilodaltons) are indicated to the left. (B) HeLa cell nuclear extract was incubated with (+) or without (−) 1 μM p21, and antibody to p62 (αp62) was used to immunopurify TFIIH from the treated or untreated extract. These immunoprecipitates were then used in kinase assays to phosphorylate GST-CTD. The positions of 32 P-labeled GST-CTD o and GST-CTD a are shown. (C) Affinity-purified cdc2 was incubated without or with p21 at various concentrations as indicated. Next, HeLa cell extract was incubated with this p21-treated cdc2. Antibody to p62 was used to immunopurify TFIIH from the cdc2-treated HeLa cell extracts. The TFIIH immunoprecipitates were then used in kinase assays to phosphorylate GST-CTD. The positions of 32 P-labeled GST-CTD o and GST-CTD a are shown.

    Journal: Molecular and Cellular Biology

    Article Title: Repression of TFIIH Transcriptional Activity and TFIIH-Associated cdk7 Kinase Activity at Mitosis

    doi:

    Figure Lengend Snippet: cdc2 inhibition of TFIIH kinase activity is reversed by the cyclin-dependent kinase inhibitor p21. (A) Affinity-purified cdc2/GST-cyclin B was incubated with the indicated concentrations of p21, and these mixtures were then used in kinase reactions with histone H1 as a substrate. The position of 32 P-labeled histone H1 is indicated. The positions of molecular mass markers (in kilodaltons) are indicated to the left. (B) HeLa cell nuclear extract was incubated with (+) or without (−) 1 μM p21, and antibody to p62 (αp62) was used to immunopurify TFIIH from the treated or untreated extract. These immunoprecipitates were then used in kinase assays to phosphorylate GST-CTD. The positions of 32 P-labeled GST-CTD o and GST-CTD a are shown. (C) Affinity-purified cdc2 was incubated without or with p21 at various concentrations as indicated. Next, HeLa cell extract was incubated with this p21-treated cdc2. Antibody to p62 was used to immunopurify TFIIH from the cdc2-treated HeLa cell extracts. The TFIIH immunoprecipitates were then used in kinase assays to phosphorylate GST-CTD. The positions of 32 P-labeled GST-CTD o and GST-CTD a are shown.

    Article Snippet: As a control, immunoprecipitates from the HeLa cell nuclear extract, prepared with an antibody to TBP, showed only very low levels of CTD phosphorylation.

    Techniques: Inhibition, Activity Assay, Affinity Purification, Incubation, Labeling

    TFIIH polypeptides are phosphorylated in vitro by mitotic kinases. HeLa cell nuclear extract (10 μg of protein per 20-μl reaction volume) was treated with mitotic kinases bound to p13-agarose beads (20 μl per reaction) and [γ- 32 P]ATP for 1 h at ambient temperature. After a brief centrifugation to remove the agarose beads, the reaction volume was brought to 100 μl with PBS and TFIIH was immunoprecipitated with protein A-Sepharose beads (25 μl per reaction) prebound with 2.5 μl of rabbit polyclonal serum to the p62 (lane 1) and ERCC3 (lane 2) subunits of TFIIH (αp62 and αERCC3, respectively) or no antibody (No Ab) (lane 3). The immunoprecipitates were washed three times with 150 μl of PBS containing 0.1% (vol/vol) Triton X-100, and precipitated proteins were eluted from the Sepharose beads with SDS sample buffer. Lane 4 shows a 1/10-volume aliquot of the labeled nuclear extract proteins (NE) prior to immunoprecipitation. Biochemically purified TFIIH (600 ng of protein) was phosphorylated with p13-bound kinase, as described above, and after removal of the kinase, TFIIH was immunoprecipitated with anti-p62 antibody (lane 5) or anti-ERCC3 antibody (lane 6) and analyzed as described above. Autophosphorylation of TFIIH is shown in lane 7. The autoradiogram of an SDS-10% PAGE gel is shown. A Western blot of the HeLa cell nuclear extract was probed with the indicated antibodies to TFIIH (anti-p62, anti-cyclin H, and anti-MO15) subunits (lanes 8 to 10, respectively). The positions of molecular mass markers (in kilodaltons) are indicated.

    Journal: Molecular and Cellular Biology

    Article Title: Repression of TFIIH Transcriptional Activity and TFIIH-Associated cdk7 Kinase Activity at Mitosis

    doi:

    Figure Lengend Snippet: TFIIH polypeptides are phosphorylated in vitro by mitotic kinases. HeLa cell nuclear extract (10 μg of protein per 20-μl reaction volume) was treated with mitotic kinases bound to p13-agarose beads (20 μl per reaction) and [γ- 32 P]ATP for 1 h at ambient temperature. After a brief centrifugation to remove the agarose beads, the reaction volume was brought to 100 μl with PBS and TFIIH was immunoprecipitated with protein A-Sepharose beads (25 μl per reaction) prebound with 2.5 μl of rabbit polyclonal serum to the p62 (lane 1) and ERCC3 (lane 2) subunits of TFIIH (αp62 and αERCC3, respectively) or no antibody (No Ab) (lane 3). The immunoprecipitates were washed three times with 150 μl of PBS containing 0.1% (vol/vol) Triton X-100, and precipitated proteins were eluted from the Sepharose beads with SDS sample buffer. Lane 4 shows a 1/10-volume aliquot of the labeled nuclear extract proteins (NE) prior to immunoprecipitation. Biochemically purified TFIIH (600 ng of protein) was phosphorylated with p13-bound kinase, as described above, and after removal of the kinase, TFIIH was immunoprecipitated with anti-p62 antibody (lane 5) or anti-ERCC3 antibody (lane 6) and analyzed as described above. Autophosphorylation of TFIIH is shown in lane 7. The autoradiogram of an SDS-10% PAGE gel is shown. A Western blot of the HeLa cell nuclear extract was probed with the indicated antibodies to TFIIH (anti-p62, anti-cyclin H, and anti-MO15) subunits (lanes 8 to 10, respectively). The positions of molecular mass markers (in kilodaltons) are indicated.

    Article Snippet: As a control, immunoprecipitates from the HeLa cell nuclear extract, prepared with an antibody to TBP, showed only very low levels of CTD phosphorylation.

    Techniques: In Vitro, Centrifugation, Immunoprecipitation, Labeling, Purification, Polyacrylamide Gel Electrophoresis, Western Blot

    Corepressor complexes implicated in TR repression contain no detectable HMT activity. (A) The corepressor complexes Sin3, SMRT, N-CoR, and Mi-2/NURD were individually immunoprecipitated from HeLa nuclear extracts with complex-specific antibodies as indicated and tested for HMT activity with core histones as substrates. Note that no detectable HMT activity was found in each immunoprecipitated complex, although HeLa nuclear extract (lane 1) contained abundant HMT activity toward H3 and less for H4. Also shown is Coomassie staining of core histones. In contrast, all immunoprecipitated corepressor complexes exhibited HDAC activity, as revealed by an in vitro deacetylation assay (B). Lane 1, immunoprecipitation with rabbit immunoglobulin G as a control for the deacetylation assay.

    Journal: Molecular and Cellular Biology

    Article Title: Involvement of Histone Methylation and Phosphorylation in Regulation of Transcription by Thyroid Hormone Receptor

    doi: 10.1128/MCB.22.16.5688-5697.2002

    Figure Lengend Snippet: Corepressor complexes implicated in TR repression contain no detectable HMT activity. (A) The corepressor complexes Sin3, SMRT, N-CoR, and Mi-2/NURD were individually immunoprecipitated from HeLa nuclear extracts with complex-specific antibodies as indicated and tested for HMT activity with core histones as substrates. Note that no detectable HMT activity was found in each immunoprecipitated complex, although HeLa nuclear extract (lane 1) contained abundant HMT activity toward H3 and less for H4. Also shown is Coomassie staining of core histones. In contrast, all immunoprecipitated corepressor complexes exhibited HDAC activity, as revealed by an in vitro deacetylation assay (B). Lane 1, immunoprecipitation with rabbit immunoglobulin G as a control for the deacetylation assay.

    Article Snippet: To assay for HMT activity in corepressor complexes of Sin3, Mi-2/NURD, SMRT, and N-CoR, the corresponding complexes were immunoprecipitated from 500 μg of HeLa nuclear extracts by incubating in 200 μl of immunoprecipitation buffer (25 mM Tris [pH 8.0], 150 mM NaCl, 1 mM DTT, 0.1% NP-40, and 1 mM PMSF) with antibodies (5 μl) specific for Sin3A (AK-11; Santa Cruz Biotechnology), CHD4 (kindly provided by Weidong Wang, National Institute on Aging), SMRT (raised against SMRT amino acids 1165 to 1363), and N-CoR (raised against N-CoR amino acids 1 to 373) , respectively.

    Techniques: HMT Assay, Activity Assay, Immunoprecipitation, Staining, In Vitro

    PKR is in splicing complexes and required for splicing and spliceosome formation. (A) β-globin pre-mRNA template was incubated in HeLa cell nuclear extract for 30 min before immunoprecipitation with NGS, αSC35 or αPKR. Western blots of PKR (top) and SC35 (bottom) are shown for soluble (Sup) and precipitated fractions (Pellet). (B) Anti-PKR Abs inhibit spliceosome formation. Labeled β-globin pre-mRNA template was incubated in HeLa cell nuclear extract for the times shown. Where indicated, αPKR cat or αPKR rbd was added 20 min before incubation. Ribonucleoprotein splicing complexes A, B, C and H were resolved by native PAGE. Left lane shows input unspliced RNA. (C - D) Recombinant PKR (rPKR) reconstitutes β-globin pre-mRNA splicing upon PKR depletion. (C) After adding NaCl to 500 mM, nuclear extract was incubated at 4 °C with αIgG or αPKR Ab for 18 h; the immunoprecipitate was removed using protein A/G Sepharose beads. The depleted extract was centrifuged through Sephadex G-25 before in vitro splicing was performed for 2 h, with rPKR or storage buffer (buffer) added as shown. (D) Plot of mRNA/pre-mRNA ratio in C ; αIgG, grey bars; αPKR, black bars. (E) In parallel, splicing of MINX RNA was assayed as in C . U, input unspliced RNA. (F) Reconstitution of spliceosomes on β-globin pre-mRNA template with rPKR. Formation of spliceosomes was assayed as in B , using extract depleted of PKR as in C . Where indicated, 480 ng of rPKR was added back. Three lanes on right show MINX RNA, assayed in parallel.

    Journal: Cell Research

    Article Title: PKR activation and eIF2α phosphorylation mediate human globin mRNA splicing at spliceosome assembly

    doi: 10.1038/cr.2017.39

    Figure Lengend Snippet: PKR is in splicing complexes and required for splicing and spliceosome formation. (A) β-globin pre-mRNA template was incubated in HeLa cell nuclear extract for 30 min before immunoprecipitation with NGS, αSC35 or αPKR. Western blots of PKR (top) and SC35 (bottom) are shown for soluble (Sup) and precipitated fractions (Pellet). (B) Anti-PKR Abs inhibit spliceosome formation. Labeled β-globin pre-mRNA template was incubated in HeLa cell nuclear extract for the times shown. Where indicated, αPKR cat or αPKR rbd was added 20 min before incubation. Ribonucleoprotein splicing complexes A, B, C and H were resolved by native PAGE. Left lane shows input unspliced RNA. (C - D) Recombinant PKR (rPKR) reconstitutes β-globin pre-mRNA splicing upon PKR depletion. (C) After adding NaCl to 500 mM, nuclear extract was incubated at 4 °C with αIgG or αPKR Ab for 18 h; the immunoprecipitate was removed using protein A/G Sepharose beads. The depleted extract was centrifuged through Sephadex G-25 before in vitro splicing was performed for 2 h, with rPKR or storage buffer (buffer) added as shown. (D) Plot of mRNA/pre-mRNA ratio in C ; αIgG, grey bars; αPKR, black bars. (E) In parallel, splicing of MINX RNA was assayed as in C . U, input unspliced RNA. (F) Reconstitution of spliceosomes on β-globin pre-mRNA template with rPKR. Formation of spliceosomes was assayed as in B , using extract depleted of PKR as in C . Where indicated, 480 ng of rPKR was added back. Three lanes on right show MINX RNA, assayed in parallel.

    Article Snippet: Immunoprecipitation from HeLa nuclear extract was performed by addition of 12 μg αPKR, 26.4 μg αSC35 (Santa Cruz) or 60 μg NGS followed by incubation for 5 h at 4 °C.

    Techniques: Incubation, Immunoprecipitation, Next-Generation Sequencing, Western Blot, Labeling, Clear Native PAGE, Recombinant, In Vitro