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  • 99
    Integrated DNA Technologies nuclease free water
    Nuclease Free Water, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 99/100, based on 483 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Worthington Biochemical micrococcal nuclease
    Micrococcal Nuclease, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 99/100, based on 1066 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher nuclease free water
    Nuclease Free Water, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 19264 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore benzonase nuclease
    HELQ interacts with the BCDX2 RAD51 paralog complex and ATR. ( a and b ) The HELQ and XRCC2 complexes were immunopurified from nuclear extracts prepared from HeLa S3 cells expressing FLAG-HA-epitope-tagged HELQ and XRCC2, respectively. The complexes were sequentially purified with anti-FLAG and anti-HA antibodies. The complexes were resolved by SDS-PAGE on a 4–20% gradient gel and visualized by silver staining. Approximate migration positions of proteins identified in gel sections are shown (left). Immunoblotting with specific antibodies confirmed the presence of the proteins in the HELQ complex (right). ( c ) BCDX2 and ATR associate with HELQ before and after MMC treatment. The FLAG-HA-tagged HELQ complex was purified from HeLa S3 with or without MMC treatment for 18 h. Similar amounts of RAD51B, C, D, XRCC2 and ATR were coimmunoprecipitated with HELQ before and after MMC treatment. Nucleic acid digestion with <t>Benzonase</t> did not influence HELQ-BCDX2 and HELQ-ATR associations. ( d ) GFP-tagged HELQ and DsRed-Monomer-tagged RAD51B were transiently coexpressed in HEK293T cells. The whole-cell extracts prepared from those transfected cells were sonicated and incubated in the presence or absence of Benzonase. GFP-tagged HELQ was immunoprecipitated with anti-GFP antibody and DsRed-Monomer-tagged RAD51B, and endogenous ATR in the immunoprecipitated samples were detected with anti-RAD51B and anti-ATR antibodies. ( e ) GFP-tagged HELQ and flag-tagged XRCC2 were transiently coexpressed in HEK293T cells. HELQ and XRCC2 association was examined with XRCC2-specific antibody after immunoprecipitation of GFP-tagged HELQ with anti-GFP antibody. Flag-tagged XRCC2 (upper bands) and endogenous XRCC2 (lower bands) were both coimmunoprecipitated with GFP-tagged HELQ. Full-size immunoblots can be found in Supplementary Fig. S5 .
    Benzonase Nuclease, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7855 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega rabbit reticulocyte lysate
    Analysis of homogeneity and translational properties of mRNAs uniformly modified with phosphorothioate moieties. (A) Schematic representation of Firefly luciferase (RNA F) in vitro transcribed (IVT) with T7 or SP6 RNA polymerase. Green dots represent phosphorothioate moieties randomly placed within the mRNA body; (B) RNA F variants co-transcriptionally 5’-capped with the cap analog (β-S-ARCA D1) and obtained in the presence of the indicated ATP:ATPαS D1 molar ratios (10:0, 9:1,2:1, 1:1, 1:4, or 0:10) are synthesized with T7 or SP6 RNA polymerase and purified on a silica membrane (NucleoSpin RNA Clean-up XS, Macherey-Nagel). The transcription yields and quality of transcripts are analyzed by electrophoresis on a 1% agarose gel. (C) Translation efficiencies are tested in <t>rabbit</t> <t>reticulocyte</t> <t>lysate</t> (Promega) using appropriate RNA F concentrations (3, 1.5, 0.75, and 0.375 ng/μL) (see Materials and Methods for further details). Translation efficiencies are determined based on measured luminescence as a function of RNA F concentration. Slopes determined for different variants of RNA F are normalized to the slope of unmodified RNA F (10:0), calculated as the mean value from three independent experiments ± standard deviation (SD). Statistical significance: * p
    Rabbit Reticulocyte Lysate, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 4958 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen nuclease free water
    Analysis of homogeneity and translational properties of mRNAs uniformly modified with phosphorothioate moieties. (A) Schematic representation of Firefly luciferase (RNA F) in vitro transcribed (IVT) with T7 or SP6 RNA polymerase. Green dots represent phosphorothioate moieties randomly placed within the mRNA body; (B) RNA F variants co-transcriptionally 5’-capped with the cap analog (β-S-ARCA D1) and obtained in the presence of the indicated ATP:ATPαS D1 molar ratios (10:0, 9:1,2:1, 1:1, 1:4, or 0:10) are synthesized with T7 or SP6 RNA polymerase and purified on a silica membrane (NucleoSpin RNA Clean-up XS, Macherey-Nagel). The transcription yields and quality of transcripts are analyzed by electrophoresis on a 1% agarose gel. (C) Translation efficiencies are tested in <t>rabbit</t> <t>reticulocyte</t> <t>lysate</t> (Promega) using appropriate RNA F concentrations (3, 1.5, 0.75, and 0.375 ng/μL) (see Materials and Methods for further details). Translation efficiencies are determined based on measured luminescence as a function of RNA F concentration. Slopes determined for different variants of RNA F are normalized to the slope of unmodified RNA F (10:0), calculated as the mean value from three independent experiments ± standard deviation (SD). Statistical significance: * p
    Nuclease Free Water, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 3425 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs micrococcal nuclease
    Analysis of homogeneity and translational properties of mRNAs uniformly modified with phosphorothioate moieties. (A) Schematic representation of Firefly luciferase (RNA F) in vitro transcribed (IVT) with T7 or SP6 RNA polymerase. Green dots represent phosphorothioate moieties randomly placed within the mRNA body; (B) RNA F variants co-transcriptionally 5’-capped with the cap analog (β-S-ARCA D1) and obtained in the presence of the indicated ATP:ATPαS D1 molar ratios (10:0, 9:1,2:1, 1:1, 1:4, or 0:10) are synthesized with T7 or SP6 RNA polymerase and purified on a silica membrane (NucleoSpin RNA Clean-up XS, Macherey-Nagel). The transcription yields and quality of transcripts are analyzed by electrophoresis on a 1% agarose gel. (C) Translation efficiencies are tested in <t>rabbit</t> <t>reticulocyte</t> <t>lysate</t> (Promega) using appropriate RNA F concentrations (3, 1.5, 0.75, and 0.375 ng/μL) (see Materials and Methods for further details). Translation efficiencies are determined based on measured luminescence as a function of RNA F concentration. Slopes determined for different variants of RNA F are normalized to the slope of unmodified RNA F (10:0), calculated as the mean value from three independent experiments ± standard deviation (SD). Statistical significance: * p
    Micrococcal Nuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2706 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega nuclease free water
    Analysis of homogeneity and translational properties of mRNAs uniformly modified with phosphorothioate moieties. (A) Schematic representation of Firefly luciferase (RNA F) in vitro transcribed (IVT) with T7 or SP6 RNA polymerase. Green dots represent phosphorothioate moieties randomly placed within the mRNA body; (B) RNA F variants co-transcriptionally 5’-capped with the cap analog (β-S-ARCA D1) and obtained in the presence of the indicated ATP:ATPαS D1 molar ratios (10:0, 9:1,2:1, 1:1, 1:4, or 0:10) are synthesized with T7 or SP6 RNA polymerase and purified on a silica membrane (NucleoSpin RNA Clean-up XS, Macherey-Nagel). The transcription yields and quality of transcripts are analyzed by electrophoresis on a 1% agarose gel. (C) Translation efficiencies are tested in <t>rabbit</t> <t>reticulocyte</t> <t>lysate</t> (Promega) using appropriate RNA F concentrations (3, 1.5, 0.75, and 0.375 ng/μL) (see Materials and Methods for further details). Translation efficiencies are determined based on measured luminescence as a function of RNA F concentration. Slopes determined for different variants of RNA F are normalized to the slope of unmodified RNA F (10:0), calculated as the mean value from three independent experiments ± standard deviation (SD). Statistical significance: * p
    Nuclease Free Water, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 3839 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore nuclease p1
    Structures and photoproducts of G-quadruplex forming sequences. A) Structures of the cis,syn and trans,anti CPDs of TT and their <t>nuclease</t> P1 degradation products. B) Known structures of human telomeric G-quadruplex forming sequences in Na + and K + solution. The nucleotide numbering system is for the Tel26 sequence, and the numbers 1-3 refer to the TTA loop numbers as shown below. C) Sequences referred to or used in this study written 5′→3′.
    Nuclease P1, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2595 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher s1 nuclease
    A Tool to Selectively Induce 8-oxoG at Telomeres (A) Schematic of the chemoptogenetic tool for inducing telomeric 8-oxoG. (B) FAP-mCer-TRF1 colocalization with telomeres in HeLaFAP clone 10 by anti-mCer immunofluorescence (IF) with telo-FISH (top panels) and mCer fluorescence with anti-RAP1 (bottom panels). (C) Immunoblot for TRF1 in whole-cell extracts from HeLaFAP. One star indicates FAP-mCer-TRF1; two stars indicate endogenous TRF1. (D) Schematic of 8-oxoG detection in telomere restriction fragments by converting 8-oxoGs to DSBs with repair enzymes and S1 nuclease. (E) S blot of telomere restriction fragments from untreated and dye + light-treated HeLaFAP cells after digestion with the indicated enzymes and S1 nuclease. (F) Quantification of the percentage of cleaved telomeric DNA (bracketed area in the gel in E) as a function of total DNA. Means ± SD of 3 independent experiments; *p
    S1 Nuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1427 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore micrococcal nuclease
    A Tool to Selectively Induce 8-oxoG at Telomeres (A) Schematic of the chemoptogenetic tool for inducing telomeric 8-oxoG. (B) FAP-mCer-TRF1 colocalization with telomeres in HeLaFAP clone 10 by anti-mCer immunofluorescence (IF) with telo-FISH (top panels) and mCer fluorescence with anti-RAP1 (bottom panels). (C) Immunoblot for TRF1 in whole-cell extracts from HeLaFAP. One star indicates FAP-mCer-TRF1; two stars indicate endogenous TRF1. (D) Schematic of 8-oxoG detection in telomere restriction fragments by converting 8-oxoGs to DSBs with repair enzymes and S1 nuclease. (E) S blot of telomere restriction fragments from untreated and dye + light-treated HeLaFAP cells after digestion with the indicated enzymes and S1 nuclease. (F) Quantification of the percentage of cleaved telomeric DNA (bracketed area in the gel in E) as a function of total DNA. Means ± SD of 3 independent experiments; *p
    Micrococcal Nuclease, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1897 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega s1 nuclease
    A template cdA induced the formation of a loop on a gapped DNA during pol β lesion bypass. Formation of a loop in the template strand of the substrates with a template dA, 5′S-cdA or 5′R-cdA in (CAG) 10 repeats opposite ( A ) or downstream ( B ) of a 1-nt gap was probed in the absence of pol β. In all panels, substrates were radiolabeled at the 5′-end of its template strand. The substrates were incubated with 0.15 units (A) or 0.05 units (B) of <t>S1</t> Nuclease at 3-, 5-, and 10-min time intervals (lanes 2–4, 7–9 and 12–14). Lanes 1, 6 and 11 represent the undigested substrate, whereas lanes 5, 10 and 15 represent size markers (M). For all the experiments, 25 nM of substrate was used. Arrows and circles indicate the major S1 Nuclease digestion products.
    S1 Nuclease, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1252 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs mung bean nuclease
    A template cdA induced the formation of a loop on a gapped DNA during pol β lesion bypass. Formation of a loop in the template strand of the substrates with a template dA, 5′S-cdA or 5′R-cdA in (CAG) 10 repeats opposite ( A ) or downstream ( B ) of a 1-nt gap was probed in the absence of pol β. In all panels, substrates were radiolabeled at the 5′-end of its template strand. The substrates were incubated with 0.15 units (A) or 0.05 units (B) of <t>S1</t> Nuclease at 3-, 5-, and 10-min time intervals (lanes 2–4, 7–9 and 12–14). Lanes 1, 6 and 11 represent the undigested substrate, whereas lanes 5, 10 and 15 represent size markers (M). For all the experiments, 25 nM of substrate was used. Arrows and circles indicate the major S1 Nuclease digestion products.
    Mung Bean Nuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1646 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa s1 nuclease
    PFGE–Southern hybridization images demonstrating  bla CMY−2  and  spvB  locations in  S . Typhimurium strains. (A)  PFGE separation of S1 nuclease-digested genomic DNA from the selected strains followed by Southern hybridization with probes specific to  bla CMY−2  and  spvB . An arrow indicates the position of the endogenous 133-kb plasmid, in which the  spvB  signal was detected. Open triangles indicate the positions of enlarged plasmids, in which both  bla CMY−2  and  spvB  signals were detected. Both signals were observed in multiple plasmid bands in strains 12-13, 12-14, and 12-18.  (B)  PFGE separation of FseI-digested genomic DNA from the selected strains followed by Southern hybridization with a  bla CMY−2  probe. An arrow indicates the original position of the restricted fragment (350 kb) in which the chromosomal  bla CMY−2  signal was detected. In the selected mutants, this fragment was not present and the  bla CMY−2  signal was identified on the largest band as indicated by a closed trangle. Open triangles indicate the position of enlarged plasmid observed in strains 12-14, 25-6, and 25-17.  (C)  PFGE separation of XbaI-digested genomic DNA from the selected strains followed by Southern hybridization with a  bla CMY−2  probe. An arrow indicates the original position of the restricted fragment (520 kb) in which chromosomal  bla CMY−2  signal was detected. Closed triangles indicate the position of  bla CMY−2  signals that originated from tandemly amplified GI-VII-6.  (D)  Restriction maps of the GI-VII-6 and its flanking region in the parent strain L-3553 and the mutants with putative amplifications. One putative amplified region presented is flanked by the first and the third copies of IS 26  (upper), and the other is flanked by the first and the fourth copies of IS 26  (lower). These amplified regions generate 100-kb (upper) and 125-kb (lower) fragments after XbaI digestion, respectively.
    S1 Nuclease, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1329 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nuclease free water
    PFGE–Southern hybridization images demonstrating  bla CMY−2  and  spvB  locations in  S . Typhimurium strains. (A)  PFGE separation of S1 nuclease-digested genomic DNA from the selected strains followed by Southern hybridization with probes specific to  bla CMY−2  and  spvB . An arrow indicates the position of the endogenous 133-kb plasmid, in which the  spvB  signal was detected. Open triangles indicate the positions of enlarged plasmids, in which both  bla CMY−2  and  spvB  signals were detected. Both signals were observed in multiple plasmid bands in strains 12-13, 12-14, and 12-18.  (B)  PFGE separation of FseI-digested genomic DNA from the selected strains followed by Southern hybridization with a  bla CMY−2  probe. An arrow indicates the original position of the restricted fragment (350 kb) in which the chromosomal  bla CMY−2  signal was detected. In the selected mutants, this fragment was not present and the  bla CMY−2  signal was identified on the largest band as indicated by a closed trangle. Open triangles indicate the position of enlarged plasmid observed in strains 12-14, 25-6, and 25-17.  (C)  PFGE separation of XbaI-digested genomic DNA from the selected strains followed by Southern hybridization with a  bla CMY−2  probe. An arrow indicates the original position of the restricted fragment (520 kb) in which chromosomal  bla CMY−2  signal was detected. Closed triangles indicate the position of  bla CMY−2  signals that originated from tandemly amplified GI-VII-6.  (D)  Restriction maps of the GI-VII-6 and its flanking region in the parent strain L-3553 and the mutants with putative amplifications. One putative amplified region presented is flanked by the first and the third copies of IS 26  (upper), and the other is flanked by the first and the fourth copies of IS 26  (lower). These amplified regions generate 100-kb (upper) and 125-kb (lower) fragments after XbaI digestion, respectively.
    Nuclease Free Water, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1067 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad nuclease free water
    PFGE–Southern hybridization images demonstrating  bla CMY−2  and  spvB  locations in  S . Typhimurium strains. (A)  PFGE separation of S1 nuclease-digested genomic DNA from the selected strains followed by Southern hybridization with probes specific to  bla CMY−2  and  spvB . An arrow indicates the position of the endogenous 133-kb plasmid, in which the  spvB  signal was detected. Open triangles indicate the positions of enlarged plasmids, in which both  bla CMY−2  and  spvB  signals were detected. Both signals were observed in multiple plasmid bands in strains 12-13, 12-14, and 12-18.  (B)  PFGE separation of FseI-digested genomic DNA from the selected strains followed by Southern hybridization with a  bla CMY−2  probe. An arrow indicates the original position of the restricted fragment (350 kb) in which the chromosomal  bla CMY−2  signal was detected. In the selected mutants, this fragment was not present and the  bla CMY−2  signal was identified on the largest band as indicated by a closed trangle. Open triangles indicate the position of enlarged plasmid observed in strains 12-14, 25-6, and 25-17.  (C)  PFGE separation of XbaI-digested genomic DNA from the selected strains followed by Southern hybridization with a  bla CMY−2  probe. An arrow indicates the original position of the restricted fragment (520 kb) in which chromosomal  bla CMY−2  signal was detected. Closed triangles indicate the position of  bla CMY−2  signals that originated from tandemly amplified GI-VII-6.  (D)  Restriction maps of the GI-VII-6 and its flanking region in the parent strain L-3553 and the mutants with putative amplifications. One putative amplified region presented is flanked by the first and the third copies of IS 26  (upper), and the other is flanked by the first and the fourth copies of IS 26  (lower). These amplified regions generate 100-kb (upper) and 125-kb (lower) fragments after XbaI digestion, respectively.
    Nuclease Free Water, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 2253 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher water nuclease free
    PFGE–Southern hybridization images demonstrating  bla CMY−2  and  spvB  locations in  S . Typhimurium strains. (A)  PFGE separation of S1 nuclease-digested genomic DNA from the selected strains followed by Southern hybridization with probes specific to  bla CMY−2  and  spvB . An arrow indicates the position of the endogenous 133-kb plasmid, in which the  spvB  signal was detected. Open triangles indicate the positions of enlarged plasmids, in which both  bla CMY−2  and  spvB  signals were detected. Both signals were observed in multiple plasmid bands in strains 12-13, 12-14, and 12-18.  (B)  PFGE separation of FseI-digested genomic DNA from the selected strains followed by Southern hybridization with a  bla CMY−2  probe. An arrow indicates the original position of the restricted fragment (350 kb) in which the chromosomal  bla CMY−2  signal was detected. In the selected mutants, this fragment was not present and the  bla CMY−2  signal was identified on the largest band as indicated by a closed trangle. Open triangles indicate the position of enlarged plasmid observed in strains 12-14, 25-6, and 25-17.  (C)  PFGE separation of XbaI-digested genomic DNA from the selected strains followed by Southern hybridization with a  bla CMY−2  probe. An arrow indicates the original position of the restricted fragment (520 kb) in which chromosomal  bla CMY−2  signal was detected. Closed triangles indicate the position of  bla CMY−2  signals that originated from tandemly amplified GI-VII-6.  (D)  Restriction maps of the GI-VII-6 and its flanking region in the parent strain L-3553 and the mutants with putative amplifications. One putative amplified region presented is flanked by the first and the third copies of IS 26  (upper), and the other is flanked by the first and the fourth copies of IS 26  (lower). These amplified regions generate 100-kb (upper) and 125-kb (lower) fragments after XbaI digestion, respectively.
    Water Nuclease Free, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher micrococcal nuclease
    PFGE–Southern hybridization images demonstrating  bla CMY−2  and  spvB  locations in  S . Typhimurium strains. (A)  PFGE separation of S1 nuclease-digested genomic DNA from the selected strains followed by Southern hybridization with probes specific to  bla CMY−2  and  spvB . An arrow indicates the position of the endogenous 133-kb plasmid, in which the  spvB  signal was detected. Open triangles indicate the positions of enlarged plasmids, in which both  bla CMY−2  and  spvB  signals were detected. Both signals were observed in multiple plasmid bands in strains 12-13, 12-14, and 12-18.  (B)  PFGE separation of FseI-digested genomic DNA from the selected strains followed by Southern hybridization with a  bla CMY−2  probe. An arrow indicates the original position of the restricted fragment (350 kb) in which the chromosomal  bla CMY−2  signal was detected. In the selected mutants, this fragment was not present and the  bla CMY−2  signal was identified on the largest band as indicated by a closed trangle. Open triangles indicate the position of enlarged plasmid observed in strains 12-14, 25-6, and 25-17.  (C)  PFGE separation of XbaI-digested genomic DNA from the selected strains followed by Southern hybridization with a  bla CMY−2  probe. An arrow indicates the original position of the restricted fragment (520 kb) in which chromosomal  bla CMY−2  signal was detected. Closed triangles indicate the position of  bla CMY−2  signals that originated from tandemly amplified GI-VII-6.  (D)  Restriction maps of the GI-VII-6 and its flanking region in the parent strain L-3553 and the mutants with putative amplifications. One putative amplified region presented is flanked by the first and the third copies of IS 26  (upper), and the other is flanked by the first and the fourth copies of IS 26  (lower). These amplified regions generate 100-kb (upper) and 125-kb (lower) fragments after XbaI digestion, respectively.
    Micrococcal Nuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 999 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore nuclease free water
    PFGE–Southern hybridization images demonstrating  bla CMY−2  and  spvB  locations in  S . Typhimurium strains. (A)  PFGE separation of S1 nuclease-digested genomic DNA from the selected strains followed by Southern hybridization with probes specific to  bla CMY−2  and  spvB . An arrow indicates the position of the endogenous 133-kb plasmid, in which the  spvB  signal was detected. Open triangles indicate the positions of enlarged plasmids, in which both  bla CMY−2  and  spvB  signals were detected. Both signals were observed in multiple plasmid bands in strains 12-13, 12-14, and 12-18.  (B)  PFGE separation of FseI-digested genomic DNA from the selected strains followed by Southern hybridization with a  bla CMY−2  probe. An arrow indicates the original position of the restricted fragment (350 kb) in which the chromosomal  bla CMY−2  signal was detected. In the selected mutants, this fragment was not present and the  bla CMY−2  signal was identified on the largest band as indicated by a closed trangle. Open triangles indicate the position of enlarged plasmid observed in strains 12-14, 25-6, and 25-17.  (C)  PFGE separation of XbaI-digested genomic DNA from the selected strains followed by Southern hybridization with a  bla CMY−2  probe. An arrow indicates the original position of the restricted fragment (520 kb) in which chromosomal  bla CMY−2  signal was detected. Closed triangles indicate the position of  bla CMY−2  signals that originated from tandemly amplified GI-VII-6.  (D)  Restriction maps of the GI-VII-6 and its flanking region in the parent strain L-3553 and the mutants with putative amplifications. One putative amplified region presented is flanked by the first and the third copies of IS 26  (upper), and the other is flanked by the first and the fourth copies of IS 26  (lower). These amplified regions generate 100-kb (upper) and 125-kb (lower) fragments after XbaI digestion, respectively.
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    Thermo Fisher cell lysis
    PFGE–Southern hybridization images demonstrating  bla CMY−2  and  spvB  locations in  S . Typhimurium strains. (A)  PFGE separation of S1 nuclease-digested genomic DNA from the selected strains followed by Southern hybridization with probes specific to  bla CMY−2  and  spvB . An arrow indicates the position of the endogenous 133-kb plasmid, in which the  spvB  signal was detected. Open triangles indicate the positions of enlarged plasmids, in which both  bla CMY−2  and  spvB  signals were detected. Both signals were observed in multiple plasmid bands in strains 12-13, 12-14, and 12-18.  (B)  PFGE separation of FseI-digested genomic DNA from the selected strains followed by Southern hybridization with a  bla CMY−2  probe. An arrow indicates the original position of the restricted fragment (350 kb) in which the chromosomal  bla CMY−2  signal was detected. In the selected mutants, this fragment was not present and the  bla CMY−2  signal was identified on the largest band as indicated by a closed trangle. Open triangles indicate the position of enlarged plasmid observed in strains 12-14, 25-6, and 25-17.  (C)  PFGE separation of XbaI-digested genomic DNA from the selected strains followed by Southern hybridization with a  bla CMY−2  probe. An arrow indicates the original position of the restricted fragment (520 kb) in which chromosomal  bla CMY−2  signal was detected. Closed triangles indicate the position of  bla CMY−2  signals that originated from tandemly amplified GI-VII-6.  (D)  Restriction maps of the GI-VII-6 and its flanking region in the parent strain L-3553 and the mutants with putative amplifications. One putative amplified region presented is flanked by the first and the third copies of IS 26  (upper), and the other is flanked by the first and the fourth copies of IS 26  (lower). These amplified regions generate 100-kb (upper) and 125-kb (lower) fragments after XbaI digestion, respectively.
    Cell Lysis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4380 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore benzonase
    RNase protection assay to detect packaging of Tf1 RNA into VLPs. Cellular extracts containing VLPs were treated with various amounts of <t>Benzonase</t> for 6 min. The RNAs extracted from the Benzonase-treated samples and from whole cells were analyzed on RNA blots. Tf1 and actin mRNA were detected by hybridization with probes specific for Tf1 Gag and actin of S. pombe . The rRNAs were visualized by ethidium bromide staining of the agarose gel under UV light. Wild Type, strain containing the wild-type sequence of Tf1; Gag-Δ, strain with deletion in the Tf1 Gag that destabilizes Gag without affecting translation of IN; T, total RNA extracted from whole cells with glass beads and phenol-chloroform; 0, 6, and 12, RNA samples extracted from the native extracts containing VLPs after treatment with 0, 6, and 12 U of Benzonase, respectively.
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    FUJIFILM nuclease p1
    UV cross-linking with the upstream cis-element or the editing site of  psbE  and  petB  mRNAs. ( A ) Schematic representation of mRNA substrates with  32 P-labels (marked by asterisks) at the center of the upstream cis-element (underlined) or at the editing site (C). ( B ) Gel patterns of proteins bound to the upstream cis-element and the editing site. ( Left )  psbE  mRNA. ( Right )  perB  mRNA. Each mRNA was incubated for 1 h at 28°C in the editing reaction mixture and then UV-irradiated (254 nm). The mixture was treated with a mixture of RNase A, nuclease P1, and  C. adamanteus  venom phosphodiesterase I followed by SDS/PAGE. Protein size markers (Rainbow, Amersham Pharmacia) are shown between the two gel patterns.
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    Thermo Fisher crispr nuclease vector
    Transforming growth factor-β (TGF-β) receptor proteins I and II (RI and RII) are core fucosylated by fucosyltransferase 8 <t>(FUT8)</t> in HEK-293 T cells. a Establishment of FUT8-knockout (KO) HEK-293 T cells by <t>CRISPR-Cas9-mediated</t> genome editing. Two independent CRISPR-Cas9 clones targeting exon 3 or 6 of FUT8 were established (upper panel). Inactivation of FUT8 gene and consequent loss of core fucosylation were validated by western blot (lower-left panel) and LCA binding assay (lower-right panel). b Protein domain organization of TGF-β RI and RII. According to the Uniprot database ( http://www.uniprot.org ), potential N-linked glycosylation sites within TGF-β RII (Asn-70, 94, and 154) and RI (Asn-45) were marked. SP, signal peptide sequence; TM, transmembrane domain. c Core fucosylation of TGF-β RI and RII proteins were eliminated by FUT8 knockout. Recombinant TGF-β RI or RII proteins in the control or two FUT8-KO 293 T cell lines were probed with biotinylated Lens culinaris lectin (LCA), then detection with streptavidin-conjugated horseradish peroxidase. kDa, kiloDalton
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    Image Search Results


    HELQ interacts with the BCDX2 RAD51 paralog complex and ATR. ( a and b ) The HELQ and XRCC2 complexes were immunopurified from nuclear extracts prepared from HeLa S3 cells expressing FLAG-HA-epitope-tagged HELQ and XRCC2, respectively. The complexes were sequentially purified with anti-FLAG and anti-HA antibodies. The complexes were resolved by SDS-PAGE on a 4–20% gradient gel and visualized by silver staining. Approximate migration positions of proteins identified in gel sections are shown (left). Immunoblotting with specific antibodies confirmed the presence of the proteins in the HELQ complex (right). ( c ) BCDX2 and ATR associate with HELQ before and after MMC treatment. The FLAG-HA-tagged HELQ complex was purified from HeLa S3 with or without MMC treatment for 18 h. Similar amounts of RAD51B, C, D, XRCC2 and ATR were coimmunoprecipitated with HELQ before and after MMC treatment. Nucleic acid digestion with Benzonase did not influence HELQ-BCDX2 and HELQ-ATR associations. ( d ) GFP-tagged HELQ and DsRed-Monomer-tagged RAD51B were transiently coexpressed in HEK293T cells. The whole-cell extracts prepared from those transfected cells were sonicated and incubated in the presence or absence of Benzonase. GFP-tagged HELQ was immunoprecipitated with anti-GFP antibody and DsRed-Monomer-tagged RAD51B, and endogenous ATR in the immunoprecipitated samples were detected with anti-RAD51B and anti-ATR antibodies. ( e ) GFP-tagged HELQ and flag-tagged XRCC2 were transiently coexpressed in HEK293T cells. HELQ and XRCC2 association was examined with XRCC2-specific antibody after immunoprecipitation of GFP-tagged HELQ with anti-GFP antibody. Flag-tagged XRCC2 (upper bands) and endogenous XRCC2 (lower bands) were both coimmunoprecipitated with GFP-tagged HELQ. Full-size immunoblots can be found in Supplementary Fig. S5 .

    Journal: Nature Communications

    Article Title: Human DNA helicase HELQ participates in DNA interstrand crosslink tolerance with ATR and RAD51 paralogs

    doi: 10.1038/ncomms3338

    Figure Lengend Snippet: HELQ interacts with the BCDX2 RAD51 paralog complex and ATR. ( a and b ) The HELQ and XRCC2 complexes were immunopurified from nuclear extracts prepared from HeLa S3 cells expressing FLAG-HA-epitope-tagged HELQ and XRCC2, respectively. The complexes were sequentially purified with anti-FLAG and anti-HA antibodies. The complexes were resolved by SDS-PAGE on a 4–20% gradient gel and visualized by silver staining. Approximate migration positions of proteins identified in gel sections are shown (left). Immunoblotting with specific antibodies confirmed the presence of the proteins in the HELQ complex (right). ( c ) BCDX2 and ATR associate with HELQ before and after MMC treatment. The FLAG-HA-tagged HELQ complex was purified from HeLa S3 with or without MMC treatment for 18 h. Similar amounts of RAD51B, C, D, XRCC2 and ATR were coimmunoprecipitated with HELQ before and after MMC treatment. Nucleic acid digestion with Benzonase did not influence HELQ-BCDX2 and HELQ-ATR associations. ( d ) GFP-tagged HELQ and DsRed-Monomer-tagged RAD51B were transiently coexpressed in HEK293T cells. The whole-cell extracts prepared from those transfected cells were sonicated and incubated in the presence or absence of Benzonase. GFP-tagged HELQ was immunoprecipitated with anti-GFP antibody and DsRed-Monomer-tagged RAD51B, and endogenous ATR in the immunoprecipitated samples were detected with anti-RAD51B and anti-ATR antibodies. ( e ) GFP-tagged HELQ and flag-tagged XRCC2 were transiently coexpressed in HEK293T cells. HELQ and XRCC2 association was examined with XRCC2-specific antibody after immunoprecipitation of GFP-tagged HELQ with anti-GFP antibody. Flag-tagged XRCC2 (upper bands) and endogenous XRCC2 (lower bands) were both coimmunoprecipitated with GFP-tagged HELQ. Full-size immunoblots can be found in Supplementary Fig. S5 .

    Article Snippet: The supernatant, nuclear extract and chromatin fractions were prepared from the cells, and the HELQ and XRCC2 complexes were immunoprecipitated from the nuclear extracts by incubating with M2 anti-FLAG agarose gel for 4 h with rotation in the presence or absence of 50 U ml−1 Benzonase Nuclease (Novagen).

    Techniques: Expressing, Purification, SDS Page, Silver Staining, Migration, Transfection, Sonication, Incubation, Immunoprecipitation, Western Blot

    Analysis of homogeneity and translational properties of mRNAs uniformly modified with phosphorothioate moieties. (A) Schematic representation of Firefly luciferase (RNA F) in vitro transcribed (IVT) with T7 or SP6 RNA polymerase. Green dots represent phosphorothioate moieties randomly placed within the mRNA body; (B) RNA F variants co-transcriptionally 5’-capped with the cap analog (β-S-ARCA D1) and obtained in the presence of the indicated ATP:ATPαS D1 molar ratios (10:0, 9:1,2:1, 1:1, 1:4, or 0:10) are synthesized with T7 or SP6 RNA polymerase and purified on a silica membrane (NucleoSpin RNA Clean-up XS, Macherey-Nagel). The transcription yields and quality of transcripts are analyzed by electrophoresis on a 1% agarose gel. (C) Translation efficiencies are tested in rabbit reticulocyte lysate (Promega) using appropriate RNA F concentrations (3, 1.5, 0.75, and 0.375 ng/μL) (see Materials and Methods for further details). Translation efficiencies are determined based on measured luminescence as a function of RNA F concentration. Slopes determined for different variants of RNA F are normalized to the slope of unmodified RNA F (10:0), calculated as the mean value from three independent experiments ± standard deviation (SD). Statistical significance: * p

    Journal: bioRxiv

    Article Title: Functional and LC-MS/MS analysis of in vitro transcribed mRNAs carrying phosphorothioate or boranophosphate moieties reveal polyA tail modifications that prevent deadenylation without compromising protein expression

    doi: 10.1101/2020.07.02.184598

    Figure Lengend Snippet: Analysis of homogeneity and translational properties of mRNAs uniformly modified with phosphorothioate moieties. (A) Schematic representation of Firefly luciferase (RNA F) in vitro transcribed (IVT) with T7 or SP6 RNA polymerase. Green dots represent phosphorothioate moieties randomly placed within the mRNA body; (B) RNA F variants co-transcriptionally 5’-capped with the cap analog (β-S-ARCA D1) and obtained in the presence of the indicated ATP:ATPαS D1 molar ratios (10:0, 9:1,2:1, 1:1, 1:4, or 0:10) are synthesized with T7 or SP6 RNA polymerase and purified on a silica membrane (NucleoSpin RNA Clean-up XS, Macherey-Nagel). The transcription yields and quality of transcripts are analyzed by electrophoresis on a 1% agarose gel. (C) Translation efficiencies are tested in rabbit reticulocyte lysate (Promega) using appropriate RNA F concentrations (3, 1.5, 0.75, and 0.375 ng/μL) (see Materials and Methods for further details). Translation efficiencies are determined based on measured luminescence as a function of RNA F concentration. Slopes determined for different variants of RNA F are normalized to the slope of unmodified RNA F (10:0), calculated as the mean value from three independent experiments ± standard deviation (SD). Statistical significance: * p

    Article Snippet: In vitro translation with rabbit reticulocyte lysate (RNA F) The Rabbit Reticulocyte Lysate System (Promega) was used to determine the influence of internal modifications on translation efficiency of RNA F. The reaction mixture (9 μL) contained: reticulocyte lysate (4 μL), amino acid mixture without leucine (0.2 μL, 1 mM solution; the final concentration in 10 μL reaction mix was 20 μM), amino acid mixture without methionine (0.2 μL, 1 mM solution; the final concentration in 10 μL reaction mix was 20 μM), potassium acetate (1.9 μL of 1 M solution), and MgCl2 (0.4 μL of 25 mM solution).

    Techniques: Modification, Luciferase, In Vitro, Synthesized, Purification, Electrophoresis, Agarose Gel Electrophoresis, Concentration Assay, Standard Deviation

    WRBcc inhibition is alleviated upon TRC40 immunodepletion. ( A ) Depletion of TRC40 was confirmed by immunoblotting. ( B ) Rabbit reticulocyte lysate was immunodepleted for TRC40 or subjected to a mock treatment. Treated lysates were then used as before for in vitro translations and post-translational translocation assays in the presence of 10 µM MBP-WRBcc. N-glycosylated forms are indicated and quantified (means ± s.e.) relative to their matched MBP control samples as before.

    Journal: Journal of Cell Science

    Article Title: TRC40 can deliver short secretory proteins to the Sec61 translocon

    doi: 10.1242/jcs.102608

    Figure Lengend Snippet: WRBcc inhibition is alleviated upon TRC40 immunodepletion. ( A ) Depletion of TRC40 was confirmed by immunoblotting. ( B ) Rabbit reticulocyte lysate was immunodepleted for TRC40 or subjected to a mock treatment. Treated lysates were then used as before for in vitro translations and post-translational translocation assays in the presence of 10 µM MBP-WRBcc. N-glycosylated forms are indicated and quantified (means ± s.e.) relative to their matched MBP control samples as before.

    Article Snippet: Nuclease-treated rabbit reticulocyte lysate for in vitro translation was from Promega.

    Techniques: Inhibition, In Vitro, Translocation Assay

    Structures and photoproducts of G-quadruplex forming sequences. A) Structures of the cis,syn and trans,anti CPDs of TT and their nuclease P1 degradation products. B) Known structures of human telomeric G-quadruplex forming sequences in Na + and K + solution. The nucleotide numbering system is for the Tel26 sequence, and the numbers 1-3 refer to the TTA loop numbers as shown below. C) Sequences referred to or used in this study written 5′→3′.

    Journal: Photochemistry and photobiology

    Article Title: Evidence for Reverse Hoogsteen Hairpin Intermediates in the Photocrosslinking of Human Telomeric DNA Sequences

    doi: 10.1111/php.12898

    Figure Lengend Snippet: Structures and photoproducts of G-quadruplex forming sequences. A) Structures of the cis,syn and trans,anti CPDs of TT and their nuclease P1 degradation products. B) Known structures of human telomeric G-quadruplex forming sequences in Na + and K + solution. The nucleotide numbering system is for the Tel26 sequence, and the numbers 1-3 refer to the TTA loop numbers as shown below. C) Sequences referred to or used in this study written 5′→3′.

    Article Snippet: Nuclease P1 (NP1) from Penicillium citrinum , was from Sigma (St. Louis, MO).

    Techniques: Sequencing

    A Tool to Selectively Induce 8-oxoG at Telomeres (A) Schematic of the chemoptogenetic tool for inducing telomeric 8-oxoG. (B) FAP-mCer-TRF1 colocalization with telomeres in HeLaFAP clone 10 by anti-mCer immunofluorescence (IF) with telo-FISH (top panels) and mCer fluorescence with anti-RAP1 (bottom panels). (C) Immunoblot for TRF1 in whole-cell extracts from HeLaFAP. One star indicates FAP-mCer-TRF1; two stars indicate endogenous TRF1. (D) Schematic of 8-oxoG detection in telomere restriction fragments by converting 8-oxoGs to DSBs with repair enzymes and S1 nuclease. (E) S blot of telomere restriction fragments from untreated and dye + light-treated HeLaFAP cells after digestion with the indicated enzymes and S1 nuclease. (F) Quantification of the percentage of cleaved telomeric DNA (bracketed area in the gel in E) as a function of total DNA. Means ± SD of 3 independent experiments; *p

    Journal: Molecular cell

    Article Title: Targeted and Persistent 8-Oxoguanine Base Damage at Telomeres Promotes Telomere Loss and Crisis

    doi: 10.1016/j.molcel.2019.04.024

    Figure Lengend Snippet: A Tool to Selectively Induce 8-oxoG at Telomeres (A) Schematic of the chemoptogenetic tool for inducing telomeric 8-oxoG. (B) FAP-mCer-TRF1 colocalization with telomeres in HeLaFAP clone 10 by anti-mCer immunofluorescence (IF) with telo-FISH (top panels) and mCer fluorescence with anti-RAP1 (bottom panels). (C) Immunoblot for TRF1 in whole-cell extracts from HeLaFAP. One star indicates FAP-mCer-TRF1; two stars indicate endogenous TRF1. (D) Schematic of 8-oxoG detection in telomere restriction fragments by converting 8-oxoGs to DSBs with repair enzymes and S1 nuclease. (E) S blot of telomere restriction fragments from untreated and dye + light-treated HeLaFAP cells after digestion with the indicated enzymes and S1 nuclease. (F) Quantification of the percentage of cleaved telomeric DNA (bracketed area in the gel in E) as a function of total DNA. Means ± SD of 3 independent experiments; *p

    Article Snippet: To calculate the percent of telomeric DNA cleaved by digestion with repair enzymes and/or S1 nuclease, the telomere signal intensity of DNA migrating below (cleaved) the intact telomeres (bulk) was divided by the total telomere intensity in the lane.

    Techniques: Immunofluorescence, Fluorescence In Situ Hybridization, Fluorescence

    A template cdA induced the formation of a loop on a gapped DNA during pol β lesion bypass. Formation of a loop in the template strand of the substrates with a template dA, 5′S-cdA or 5′R-cdA in (CAG) 10 repeats opposite ( A ) or downstream ( B ) of a 1-nt gap was probed in the absence of pol β. In all panels, substrates were radiolabeled at the 5′-end of its template strand. The substrates were incubated with 0.15 units (A) or 0.05 units (B) of S1 Nuclease at 3-, 5-, and 10-min time intervals (lanes 2–4, 7–9 and 12–14). Lanes 1, 6 and 11 represent the undigested substrate, whereas lanes 5, 10 and 15 represent size markers (M). For all the experiments, 25 nM of substrate was used. Arrows and circles indicate the major S1 Nuclease digestion products.

    Journal: Nucleic Acids Research

    Article Title: A 5′, 8-cyclo-2′-deoxypurine lesion induces trinucleotide repeat deletion via a unique lesion bypass by DNA polymerase β

    doi: 10.1093/nar/gku1239

    Figure Lengend Snippet: A template cdA induced the formation of a loop on a gapped DNA during pol β lesion bypass. Formation of a loop in the template strand of the substrates with a template dA, 5′S-cdA or 5′R-cdA in (CAG) 10 repeats opposite ( A ) or downstream ( B ) of a 1-nt gap was probed in the absence of pol β. In all panels, substrates were radiolabeled at the 5′-end of its template strand. The substrates were incubated with 0.15 units (A) or 0.05 units (B) of S1 Nuclease at 3-, 5-, and 10-min time intervals (lanes 2–4, 7–9 and 12–14). Lanes 1, 6 and 11 represent the undigested substrate, whereas lanes 5, 10 and 15 represent size markers (M). For all the experiments, 25 nM of substrate was used. Arrows and circles indicate the major S1 Nuclease digestion products.

    Article Snippet: To test this possibility, we initially examined the formation of a hairpin/loop in an intact double-strand DNA using S1 Nuclease, a single strand DNA specific nuclease ( ) (Supplementary Figure S1).

    Techniques: Incubation

    PFGE–Southern hybridization images demonstrating  bla CMY−2  and  spvB  locations in  S . Typhimurium strains. (A)  PFGE separation of S1 nuclease-digested genomic DNA from the selected strains followed by Southern hybridization with probes specific to  bla CMY−2  and  spvB . An arrow indicates the position of the endogenous 133-kb plasmid, in which the  spvB  signal was detected. Open triangles indicate the positions of enlarged plasmids, in which both  bla CMY−2  and  spvB  signals were detected. Both signals were observed in multiple plasmid bands in strains 12-13, 12-14, and 12-18.  (B)  PFGE separation of FseI-digested genomic DNA from the selected strains followed by Southern hybridization with a  bla CMY−2  probe. An arrow indicates the original position of the restricted fragment (350 kb) in which the chromosomal  bla CMY−2  signal was detected. In the selected mutants, this fragment was not present and the  bla CMY−2  signal was identified on the largest band as indicated by a closed trangle. Open triangles indicate the position of enlarged plasmid observed in strains 12-14, 25-6, and 25-17.  (C)  PFGE separation of XbaI-digested genomic DNA from the selected strains followed by Southern hybridization with a  bla CMY−2  probe. An arrow indicates the original position of the restricted fragment (520 kb) in which chromosomal  bla CMY−2  signal was detected. Closed triangles indicate the position of  bla CMY−2  signals that originated from tandemly amplified GI-VII-6.  (D)  Restriction maps of the GI-VII-6 and its flanking region in the parent strain L-3553 and the mutants with putative amplifications. One putative amplified region presented is flanked by the first and the third copies of IS 26  (upper), and the other is flanked by the first and the fourth copies of IS 26  (lower). These amplified regions generate 100-kb (upper) and 125-kb (lower) fragments after XbaI digestion, respectively.

    Journal: Frontiers in Microbiology

    Article Title: Extensive amplification of GI-VII-6, a multidrug resistance genomic island of Salmonella enterica serovar Typhimurium, increases resistance to extended-spectrum cephalosporins

    doi: 10.3389/fmicb.2015.00078

    Figure Lengend Snippet: PFGE–Southern hybridization images demonstrating bla CMY−2 and spvB locations in S . Typhimurium strains. (A) PFGE separation of S1 nuclease-digested genomic DNA from the selected strains followed by Southern hybridization with probes specific to bla CMY−2 and spvB . An arrow indicates the position of the endogenous 133-kb plasmid, in which the spvB signal was detected. Open triangles indicate the positions of enlarged plasmids, in which both bla CMY−2 and spvB signals were detected. Both signals were observed in multiple plasmid bands in strains 12-13, 12-14, and 12-18. (B) PFGE separation of FseI-digested genomic DNA from the selected strains followed by Southern hybridization with a bla CMY−2 probe. An arrow indicates the original position of the restricted fragment (350 kb) in which the chromosomal bla CMY−2 signal was detected. In the selected mutants, this fragment was not present and the bla CMY−2 signal was identified on the largest band as indicated by a closed trangle. Open triangles indicate the position of enlarged plasmid observed in strains 12-14, 25-6, and 25-17. (C) PFGE separation of XbaI-digested genomic DNA from the selected strains followed by Southern hybridization with a bla CMY−2 probe. An arrow indicates the original position of the restricted fragment (520 kb) in which chromosomal bla CMY−2 signal was detected. Closed triangles indicate the position of bla CMY−2 signals that originated from tandemly amplified GI-VII-6. (D) Restriction maps of the GI-VII-6 and its flanking region in the parent strain L-3553 and the mutants with putative amplifications. One putative amplified region presented is flanked by the first and the third copies of IS 26 (upper), and the other is flanked by the first and the fourth copies of IS 26 (lower). These amplified regions generate 100-kb (upper) and 125-kb (lower) fragments after XbaI digestion, respectively.

    Article Snippet: The plugs were digested with 60 U/ml of XbaI (Takara Bio Inc.) or 40 U/ml of FseI (New England BioLabs, Ipswich, MA, USA) at 37°C for 6 h, or 2 U/ml of S1 nuclease (Takara Bio Inc.) at 37°C for 45 min. Primers used for probe synthesis were listed in Table .

    Techniques: Hybridization, Plasmid Preparation, Amplification

    RNase protection assay to detect packaging of Tf1 RNA into VLPs. Cellular extracts containing VLPs were treated with various amounts of Benzonase for 6 min. The RNAs extracted from the Benzonase-treated samples and from whole cells were analyzed on RNA blots. Tf1 and actin mRNA were detected by hybridization with probes specific for Tf1 Gag and actin of S. pombe . The rRNAs were visualized by ethidium bromide staining of the agarose gel under UV light. Wild Type, strain containing the wild-type sequence of Tf1; Gag-Δ, strain with deletion in the Tf1 Gag that destabilizes Gag without affecting translation of IN; T, total RNA extracted from whole cells with glass beads and phenol-chloroform; 0, 6, and 12, RNA samples extracted from the native extracts containing VLPs after treatment with 0, 6, and 12 U of Benzonase, respectively.

    Journal: Molecular and Cellular Biology

    Article Title: Reverse Transcription of a Self-Primed Retrotransposon Requires an RNA Structure Similar to the U5-IR Stem-Loop of Retroviruses

    doi:

    Figure Lengend Snippet: RNase protection assay to detect packaging of Tf1 RNA into VLPs. Cellular extracts containing VLPs were treated with various amounts of Benzonase for 6 min. The RNAs extracted from the Benzonase-treated samples and from whole cells were analyzed on RNA blots. Tf1 and actin mRNA were detected by hybridization with probes specific for Tf1 Gag and actin of S. pombe . The rRNAs were visualized by ethidium bromide staining of the agarose gel under UV light. Wild Type, strain containing the wild-type sequence of Tf1; Gag-Δ, strain with deletion in the Tf1 Gag that destabilizes Gag without affecting translation of IN; T, total RNA extracted from whole cells with glass beads and phenol-chloroform; 0, 6, and 12, RNA samples extracted from the native extracts containing VLPs after treatment with 0, 6, and 12 U of Benzonase, respectively.

    Article Snippet: Supernatants of the cell extracts were recovered after centrifugation at 1,000 × g for 5 min. Fifteen microliters of 10× buffer M (100 mM Tris-HCl [pH 7.5], 100 mM MgCl2 , 500 mM NaCl) with or without Benzonase (Sigma catalog no. E-8263) at specified concentrations was added to 135 μl of the supernatant and incubated at room temperature for 6 min.

    Techniques: Rnase Protection Assay, Hybridization, Staining, Agarose Gel Electrophoresis, Sequencing

    UV cross-linking with the upstream cis-element or the editing site of  psbE  and  petB  mRNAs. ( A ) Schematic representation of mRNA substrates with  32 P-labels (marked by asterisks) at the center of the upstream cis-element (underlined) or at the editing site (C). ( B ) Gel patterns of proteins bound to the upstream cis-element and the editing site. ( Left )  psbE  mRNA. ( Right )  perB  mRNA. Each mRNA was incubated for 1 h at 28°C in the editing reaction mixture and then UV-irradiated (254 nm). The mixture was treated with a mixture of RNase A, nuclease P1, and  C. adamanteus  venom phosphodiesterase I followed by SDS/PAGE. Protein size markers (Rainbow, Amersham Pharmacia) are shown between the two gel patterns.

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

    Article Title: A site-specific factor interacts directly with its cognate RNA editing site in chloroplast transcripts

    doi: 10.1073/pnas.0307163101

    Figure Lengend Snippet: UV cross-linking with the upstream cis-element or the editing site of psbE and petB mRNAs. ( A ) Schematic representation of mRNA substrates with 32 P-labels (marked by asterisks) at the center of the upstream cis-element (underlined) or at the editing site (C). ( B ) Gel patterns of proteins bound to the upstream cis-element and the editing site. ( Left ) psbE mRNA. ( Right ) perB mRNA. Each mRNA was incubated for 1 h at 28°C in the editing reaction mixture and then UV-irradiated (254 nm). The mixture was treated with a mixture of RNase A, nuclease P1, and C. adamanteus venom phosphodiesterase I followed by SDS/PAGE. Protein size markers (Rainbow, Amersham Pharmacia) are shown between the two gel patterns.

    Article Snippet: For RNA editing assays, an mRNA substrate was incubated at 28°C for 2 h. RNA was isolated and digested into 5′ mononucleotides with 1 μg of nuclease P1 (Wako Pure Chemical, Osaka) in the presence of 50 mM ammonium acetate (pH 4.8) at 37°C for 3 h. Mononucleotides were separated on cellulose TLC plates (20 × 20 cm, Funakoshi, Tokyo) by using isopropyl alcohol/HCl/water (70:15:15).

    Techniques: Incubation, Irradiation, SDS Page

    Transforming growth factor-β (TGF-β) receptor proteins I and II (RI and RII) are core fucosylated by fucosyltransferase 8 (FUT8) in HEK-293 T cells. a Establishment of FUT8-knockout (KO) HEK-293 T cells by CRISPR-Cas9-mediated genome editing. Two independent CRISPR-Cas9 clones targeting exon 3 or 6 of FUT8 were established (upper panel). Inactivation of FUT8 gene and consequent loss of core fucosylation were validated by western blot (lower-left panel) and LCA binding assay (lower-right panel). b Protein domain organization of TGF-β RI and RII. According to the Uniprot database ( http://www.uniprot.org ), potential N-linked glycosylation sites within TGF-β RII (Asn-70, 94, and 154) and RI (Asn-45) were marked. SP, signal peptide sequence; TM, transmembrane domain. c Core fucosylation of TGF-β RI and RII proteins were eliminated by FUT8 knockout. Recombinant TGF-β RI or RII proteins in the control or two FUT8-KO 293 T cell lines were probed with biotinylated Lens culinaris lectin (LCA), then detection with streptavidin-conjugated horseradish peroxidase. kDa, kiloDalton

    Journal: Breast Cancer Research : BCR

    Article Title: FUT8 promotes breast cancer cell invasiveness by remodeling TGF-β receptor core fucosylation

    doi: 10.1186/s13058-017-0904-8

    Figure Lengend Snippet: Transforming growth factor-β (TGF-β) receptor proteins I and II (RI and RII) are core fucosylated by fucosyltransferase 8 (FUT8) in HEK-293 T cells. a Establishment of FUT8-knockout (KO) HEK-293 T cells by CRISPR-Cas9-mediated genome editing. Two independent CRISPR-Cas9 clones targeting exon 3 or 6 of FUT8 were established (upper panel). Inactivation of FUT8 gene and consequent loss of core fucosylation were validated by western blot (lower-left panel) and LCA binding assay (lower-right panel). b Protein domain organization of TGF-β RI and RII. According to the Uniprot database ( http://www.uniprot.org ), potential N-linked glycosylation sites within TGF-β RII (Asn-70, 94, and 154) and RI (Asn-45) were marked. SP, signal peptide sequence; TM, transmembrane domain. c Core fucosylation of TGF-β RI and RII proteins were eliminated by FUT8 knockout. Recombinant TGF-β RI or RII proteins in the control or two FUT8-KO 293 T cell lines were probed with biotinylated Lens culinaris lectin (LCA), then detection with streptavidin-conjugated horseradish peroxidase. kDa, kiloDalton

    Article Snippet: CRISPR/Cas9-mediated genome editing To generate FUT8 -knockout (FUT8-KO) cells with the CRISPR/Cas9 system, guide RNAs (gRNAs) targeting the human FUT8 gene at exon 3 or 6 were cloned into the GeneArt CRISPR Nuclease Vector (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Knock-Out, CRISPR, Clone Assay, Western Blot, Binding Assay, Sequencing, Recombinant

    Fucosyltransferase 8 (FUT8) knockout impairs transforming growth factor-β1 (TGF) ligand binding and decreases TGF-β downstream signaling in MDA-MB-231 cells. a Establishment of FUT8-knockout (KO) MDA-MB-231 cells by CRISPR-Cas9-mediated genome editing. Two independent KO #1 and #2 clones targeting exon 3 or 6 of FUT8 were established (upper panel). The deletion of FUT8 and loss of core fucosylation were confirmed by western blot (lower-left panel) and LCA binding assay (lower-right panel) in MDA-MB-231 cells. Of note, FUT8 inactivation did not affect the expression of TGF-β RI or RII protein (lower-left panel). b Core fucosylation of TGF-β RI and RII protein were eliminated by FUT8 KO. Recombinant TGF-β RI and RII proteins produced in the control or two FUT8-KO MDA-MB-231 cell lines were probed with biotinylated Lens culinaris lectin (LCA), then detected by streptavidin-conjugated horseradish peroxidase. c Reporter assay with a TGF-β1-responsive luciferase reporter, 3TP-lux, showed significantly reduced TGF-β1-mediated signaling in FUT8-KO cells, which was rescued completely by FUT8 overexpression. d Schematic figure showing the indirect ligand binding assay. HEK-293 T cells were transfected with empty vector or the expression plasmid encoding HIS-tagged TGF-β1 (HIS.TGF-β1) protein. After 2 days, conditioned medium were added to MDA-MB-231 cells endogenously expressing TGF-β receptors. Bound HIS.TGF-β1 protein was quantified by the alkaline phosphatase (AP)-conjugated anti-HIS antibody reacting with its colorimetric substrate p-nitrophenyl phosphate. e FUT8 knockout reduced TGF-β1 ligand binding. Data are mean ± SD. * P

    Journal: Breast Cancer Research : BCR

    Article Title: FUT8 promotes breast cancer cell invasiveness by remodeling TGF-β receptor core fucosylation

    doi: 10.1186/s13058-017-0904-8

    Figure Lengend Snippet: Fucosyltransferase 8 (FUT8) knockout impairs transforming growth factor-β1 (TGF) ligand binding and decreases TGF-β downstream signaling in MDA-MB-231 cells. a Establishment of FUT8-knockout (KO) MDA-MB-231 cells by CRISPR-Cas9-mediated genome editing. Two independent KO #1 and #2 clones targeting exon 3 or 6 of FUT8 were established (upper panel). The deletion of FUT8 and loss of core fucosylation were confirmed by western blot (lower-left panel) and LCA binding assay (lower-right panel) in MDA-MB-231 cells. Of note, FUT8 inactivation did not affect the expression of TGF-β RI or RII protein (lower-left panel). b Core fucosylation of TGF-β RI and RII protein were eliminated by FUT8 KO. Recombinant TGF-β RI and RII proteins produced in the control or two FUT8-KO MDA-MB-231 cell lines were probed with biotinylated Lens culinaris lectin (LCA), then detected by streptavidin-conjugated horseradish peroxidase. c Reporter assay with a TGF-β1-responsive luciferase reporter, 3TP-lux, showed significantly reduced TGF-β1-mediated signaling in FUT8-KO cells, which was rescued completely by FUT8 overexpression. d Schematic figure showing the indirect ligand binding assay. HEK-293 T cells were transfected with empty vector or the expression plasmid encoding HIS-tagged TGF-β1 (HIS.TGF-β1) protein. After 2 days, conditioned medium were added to MDA-MB-231 cells endogenously expressing TGF-β receptors. Bound HIS.TGF-β1 protein was quantified by the alkaline phosphatase (AP)-conjugated anti-HIS antibody reacting with its colorimetric substrate p-nitrophenyl phosphate. e FUT8 knockout reduced TGF-β1 ligand binding. Data are mean ± SD. * P

    Article Snippet: CRISPR/Cas9-mediated genome editing To generate FUT8 -knockout (FUT8-KO) cells with the CRISPR/Cas9 system, guide RNAs (gRNAs) targeting the human FUT8 gene at exon 3 or 6 were cloned into the GeneArt CRISPR Nuclease Vector (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Knock-Out, Ligand Binding Assay, Multiple Displacement Amplification, CRISPR, Clone Assay, Western Blot, Binding Assay, Expressing, Recombinant, Produced, Reporter Assay, Luciferase, Over Expression, Transfection, Plasmid Preparation