Structured Review

TaKaRa open reading frame orf
Open Reading Frame Orf, supplied by TaKaRa, used in various techniques. Bioz Stars score: 89/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 89 stars, based on 3 article reviews
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open reading frame orf - by Bioz Stars, 2020-09
89/100 stars

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Polymerase Chain Reaction:

Article Title: In Vivo Study of the HC-TN Strain of Hepatitis C Virus Recovered from a Patient with Fulminant Hepatitis: RNA Transcripts of a Molecular Clone (pHC-TN) Are Infectious in Chimpanzees but Not in Huh7.5 Cells ▿
Article Snippet: .. To clone the entire open reading frame (ORF), we performed a one-round long PCR using an Advantage PCR polymerase mix (Clontech, Palo Alto, CA) ( ). .. Gel-purified amplicons were A tailed with Taq DNA polymerase (Life Technologies) at 72°C for 1 h and cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA).

Clone Assay:

Article Title: Identification of a novel nuclear localization signal and speckle-targeting sequence of tuftelin-interacting protein 11, a splicing factor involved in spliceosome disassembly
Article Snippet: .. As previously reported [ ], mouse TFIP11 cDNA corresponding to the entire open-reading frame (ORF) of 838 amino acids minus the initial ATG was cloned into the vector pEGFP-C1 (Clontech, Mountain View, CA) and the resulting plasmid was named TFIP11-C1 ( ). .. All C-terminal deletions were created in an identical manner using PCR in which the reverse primer ended as the coding sequence indicated, and this was immediately followed by a stop codon.

Plasmid Preparation:

Article Title: Identification of a novel nuclear localization signal and speckle-targeting sequence of tuftelin-interacting protein 11, a splicing factor involved in spliceosome disassembly
Article Snippet: .. As previously reported [ ], mouse TFIP11 cDNA corresponding to the entire open-reading frame (ORF) of 838 amino acids minus the initial ATG was cloned into the vector pEGFP-C1 (Clontech, Mountain View, CA) and the resulting plasmid was named TFIP11-C1 ( ). .. All C-terminal deletions were created in an identical manner using PCR in which the reverse primer ended as the coding sequence indicated, and this was immediately followed by a stop codon.

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  • 85
    TaKaRa affinity purified p32 polyclonal antiserum
    The ORF 73 protein interacts with the cellular protein <t>p32.</t> (a) Control GST alone and GST-ORF 73 were expressed in E. coli and purified from crude lysate by incubation with glutathione-Sepharose 4B affinity beads. Protein extracts of untransfected cells were incubated with GST or GST-ORF 73 proteins. Bound proteins were resolved by SDS-PAGE (12% polyacrylamide), and p32 was detected by Western blotting using p32 antiserum. A control lane of whole-cell lysate is shown to estimate the level of interaction between p32 and GST-73. (b) To confirm the expression of GST and GST-ORF 73 proteins, Western blot analysis was performed using a mouse monoclonal GST antibody. (c) Mock-infected (Uni), HVS-infected, pcDNAGFP-transfected, or p73GFP-transfected cell extracts were incubated with ORF 73 <t>polyclonal</t> antiserum bound to protein A-Sepharose beads. The beads were then pelleted and washed, and precipitated polypeptides were resolved on an SDS-12% polyacrylamide gel and analyzed by immunoblot analysis with p32 antiserum. (d) Mock-infected OMK (Uni) and HVS-infected cell extracts were incubated with rp32-Sepharose (i) or rGO-Sepharose (ii). After being washed and eluted, the cells were separated by SDS-PAGE and analyzed by Western blotting with ORF 73 polyclonal antisera. (e) Untransfected Cos-7 and pcDNAGFP-transfected or p73GFP-transfected cell extracts were incubated with rp32-Sepharose (i) or rGO-Sepharose (ii). After being washed and eluted, the cells were separated by SDS-PAGE and analyzed by Western blotting with GFP monoclonal antisera.
    Affinity Purified P32 Polyclonal Antiserum, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/affinity purified p32 polyclonal antiserum/product/TaKaRa
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
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    85
    TaKaRa flag hsnm1 open reading frame
    <t>hSnm1</t> focus formation occurs normally in Nijmegen breakage syndrome cells. GM7166 primary fibroblasts were fixed 9 h after mock treatment or after exposure to 15 Gray of ionizing radiation (IR) and subjected to indirect immunofluorescence with anti-hSnm1. (a) Mock-treated cells; (b) after ionizing radiation exposure.
    Flag Hsnm1 Open Reading Frame, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/flag hsnm1 open reading frame/product/TaKaRa
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    Price from $9.99 to $1999.99
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    85
    TaKaRa c pomonella
    The full-length cDNA sequence of Cydia <t>pomonella</t> CYP9A61 and deduced amino acid sequence. The start codon, stop codon, and polyadenylation signal sequences are underlined. The heme-binding domain and six substrate recognition sites (SRSs) are boxed. Dotted lines indicate the domains on which degenerate primers were designed.
    C Pomonella, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/c pomonella/product/TaKaRa
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    85
    TaKaRa atxn8os cdna
    IRES activity of the <t>ATXN8OS</t> transcript. (A) ATXN8OS organization with promoter (open arrow), exons (open boxes) and functional splice donor sequences (GT) of D exons (D5, D4, D, D″ and D′) indicated. The CTG repeat tract is located in exon A. Transcription start site of exon D5 and exon D are represented by +1 and +801, respectively. (B) ATXN8OS RNA (NR_002717) generated from the splicing events represented by the wavy lines. The putative ORF initiated from AUG +1247 is indicated by the open boxes inside the RNA. The restriction enzymes and the cutting sites used to generate +801∼+1195 <t>cDNA</t> fragment of ATXN8OS are shown on the bottom of the cDNA. (C) The dual luciferase reporter plasmid had Renilla luciferase and firefly luciferase genes between the TK promoter and polyadenylation signal. The locations of Xba I, Xho I and Bam HI sites used for construction are shown on the top. (D) Relative luciferase activities generated by dual luciferase constructs with ECMV IRES and ATXN8OS +801∼+1195 cDNA fragment in HEK-293 and IMR-32 cells. Forty-eight hours following transfection, cells were harvested and luciferases activities were measured. IRES activity is expressed as percentages of the activity of the ECMV IRES, which was set at 100%. In addition, relative luciferase activities with ATXN8OS +801∼+953 and +953∼+1195 cDNA fragments were measured in HEK-293 cells, with IRES activity of +801∼+1195 set at 100%. Each value is the mean ± SD of three independent experiments each performed in duplicate.
    Atxn8os Cdna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The ORF 73 protein interacts with the cellular protein p32. (a) Control GST alone and GST-ORF 73 were expressed in E. coli and purified from crude lysate by incubation with glutathione-Sepharose 4B affinity beads. Protein extracts of untransfected cells were incubated with GST or GST-ORF 73 proteins. Bound proteins were resolved by SDS-PAGE (12% polyacrylamide), and p32 was detected by Western blotting using p32 antiserum. A control lane of whole-cell lysate is shown to estimate the level of interaction between p32 and GST-73. (b) To confirm the expression of GST and GST-ORF 73 proteins, Western blot analysis was performed using a mouse monoclonal GST antibody. (c) Mock-infected (Uni), HVS-infected, pcDNAGFP-transfected, or p73GFP-transfected cell extracts were incubated with ORF 73 polyclonal antiserum bound to protein A-Sepharose beads. The beads were then pelleted and washed, and precipitated polypeptides were resolved on an SDS-12% polyacrylamide gel and analyzed by immunoblot analysis with p32 antiserum. (d) Mock-infected OMK (Uni) and HVS-infected cell extracts were incubated with rp32-Sepharose (i) or rGO-Sepharose (ii). After being washed and eluted, the cells were separated by SDS-PAGE and analyzed by Western blotting with ORF 73 polyclonal antisera. (e) Untransfected Cos-7 and pcDNAGFP-transfected or p73GFP-transfected cell extracts were incubated with rp32-Sepharose (i) or rGO-Sepharose (ii). After being washed and eluted, the cells were separated by SDS-PAGE and analyzed by Western blotting with GFP monoclonal antisera.

    Journal: Journal of Virology

    Article Title: The Herpesvirus Saimiri Open Reading Frame 73 Gene Product Interacts with the Cellular Protein p32

    doi: 10.1128/JVI.76.22.11612-11622.2002

    Figure Lengend Snippet: The ORF 73 protein interacts with the cellular protein p32. (a) Control GST alone and GST-ORF 73 were expressed in E. coli and purified from crude lysate by incubation with glutathione-Sepharose 4B affinity beads. Protein extracts of untransfected cells were incubated with GST or GST-ORF 73 proteins. Bound proteins were resolved by SDS-PAGE (12% polyacrylamide), and p32 was detected by Western blotting using p32 antiserum. A control lane of whole-cell lysate is shown to estimate the level of interaction between p32 and GST-73. (b) To confirm the expression of GST and GST-ORF 73 proteins, Western blot analysis was performed using a mouse monoclonal GST antibody. (c) Mock-infected (Uni), HVS-infected, pcDNAGFP-transfected, or p73GFP-transfected cell extracts were incubated with ORF 73 polyclonal antiserum bound to protein A-Sepharose beads. The beads were then pelleted and washed, and precipitated polypeptides were resolved on an SDS-12% polyacrylamide gel and analyzed by immunoblot analysis with p32 antiserum. (d) Mock-infected OMK (Uni) and HVS-infected cell extracts were incubated with rp32-Sepharose (i) or rGO-Sepharose (ii). After being washed and eluted, the cells were separated by SDS-PAGE and analyzed by Western blotting with ORF 73 polyclonal antisera. (e) Untransfected Cos-7 and pcDNAGFP-transfected or p73GFP-transfected cell extracts were incubated with rp32-Sepharose (i) or rGO-Sepharose (ii). After being washed and eluted, the cells were separated by SDS-PAGE and analyzed by Western blotting with GFP monoclonal antisera.

    Article Snippet: The membranes were then incubated with either a 1:400 dilution of affinity-purified p32 polyclonal antiserum , a 1:500 dilution of monoclonal p32 antiserum , a 1:1,000 dilution of green fluorescent protein monoclonal antiserum (Clontech), or a 1:2,000 dilution of GST monoclonal antiserum (Sigma), washed with PBS, and incubated for 1 h at 37°C with a 1:1,000 dilution of anti-rabbit or anti-mouse immunoglobulin conjugated with horseradish peroxidase (Dako) in blocking buffer.

    Techniques: Purification, Incubation, SDS Page, Western Blot, Expressing, Infection, Transfection

    The ORF 73 protein accumulates p32 in the nucleus. In all color images, ORF73-GFP or ORF 73 in infected cells stains green, 4; 6-diamidino-2-phenylindole (DAPI) stains blue, and p32 stains red. Images represent a single 0.36-μm focal plane approximately halfway through the cell. Bars, 10 μm. Cells were transfected with p73GFP (i and v), infected with HVS (viii), or transfected with p73ΔNLS1GFP (xi). Each experiment resulted in nuclear localization of ORF73 (or the GFP fusion protein), as shown by DAPI costaining (ii and xii). For infected cells, ORF73 was detected by ORF73 polyclonal antiserum and an anti-rabbit fluorescein thiocyanate conjugate (viii). In all cases, p32 was detected using an p32 antiserum (Santa Cruz) and an anti-goat Texas red conjugate (iii, vi, ix, and xiii). Images iv, vii, x, and xiv are an overlay of the three channels (i to iii), (v to vi), (viii to ix), and (xi to xiii), respectively.

    Journal: Journal of Virology

    Article Title: The Herpesvirus Saimiri Open Reading Frame 73 Gene Product Interacts with the Cellular Protein p32

    doi: 10.1128/JVI.76.22.11612-11622.2002

    Figure Lengend Snippet: The ORF 73 protein accumulates p32 in the nucleus. In all color images, ORF73-GFP or ORF 73 in infected cells stains green, 4; 6-diamidino-2-phenylindole (DAPI) stains blue, and p32 stains red. Images represent a single 0.36-μm focal plane approximately halfway through the cell. Bars, 10 μm. Cells were transfected with p73GFP (i and v), infected with HVS (viii), or transfected with p73ΔNLS1GFP (xi). Each experiment resulted in nuclear localization of ORF73 (or the GFP fusion protein), as shown by DAPI costaining (ii and xii). For infected cells, ORF73 was detected by ORF73 polyclonal antiserum and an anti-rabbit fluorescein thiocyanate conjugate (viii). In all cases, p32 was detected using an p32 antiserum (Santa Cruz) and an anti-goat Texas red conjugate (iii, vi, ix, and xiii). Images iv, vii, x, and xiv are an overlay of the three channels (i to iii), (v to vi), (viii to ix), and (xi to xiii), respectively.

    Article Snippet: The membranes were then incubated with either a 1:400 dilution of affinity-purified p32 polyclonal antiserum , a 1:500 dilution of monoclonal p32 antiserum , a 1:1,000 dilution of green fluorescent protein monoclonal antiserum (Clontech), or a 1:2,000 dilution of GST monoclonal antiserum (Sigma), washed with PBS, and incubated for 1 h at 37°C with a 1:1,000 dilution of anti-rabbit or anti-mouse immunoglobulin conjugated with horseradish peroxidase (Dako) in blocking buffer.

    Techniques: Infection, Transfection

    hSnm1 focus formation occurs normally in Nijmegen breakage syndrome cells. GM7166 primary fibroblasts were fixed 9 h after mock treatment or after exposure to 15 Gray of ionizing radiation (IR) and subjected to indirect immunofluorescence with anti-hSnm1. (a) Mock-treated cells; (b) after ionizing radiation exposure.

    Journal: Molecular and Cellular Biology

    Article Title: hSnm1 Colocalizes and Physically Associates with 53BP1 before and after DNA Damage

    doi: 10.1128/MCB.22.24.8635-8647.2002

    Figure Lengend Snippet: hSnm1 focus formation occurs normally in Nijmegen breakage syndrome cells. GM7166 primary fibroblasts were fixed 9 h after mock treatment or after exposure to 15 Gray of ionizing radiation (IR) and subjected to indirect immunofluorescence with anti-hSnm1. (a) Mock-treated cells; (b) after ionizing radiation exposure.

    Article Snippet: The Flag-hSnm1 open reading frame was subsequently cloned into the mammalian expression vector pEBS7 ( ) and also fused with enhanced green fluorescent protein (EGFP) with the pEGFP-C1 vector from Clontech.

    Techniques: Immunofluorescence

    Colocalization of hSnm1 foci with hMre11 and BRCA1 but not hRad51, as determined by indirect immunofluorescence. HT-1080 cells were irradiated with 10 Gray and stained 2 h later with (a) anti-hSnm1 and (b) anti-hRad51; (c) merged fields after DAPI staining. HT-1080 cells were irradiated with 15 Gray and stained 9 h later with (d) anti-hSnm1 and (e) anti-hMre11; (f) merged fields after DAPI staining. Partial colocalization with BRCA1 ionizing radiation-induced foci was observed 5 h after 10 Gray, as shown by (g) anti-hSnm1 and (h) anti-BRCA1; (i) both fields merged with DAPI staining.

    Journal: Molecular and Cellular Biology

    Article Title: hSnm1 Colocalizes and Physically Associates with 53BP1 before and after DNA Damage

    doi: 10.1128/MCB.22.24.8635-8647.2002

    Figure Lengend Snippet: Colocalization of hSnm1 foci with hMre11 and BRCA1 but not hRad51, as determined by indirect immunofluorescence. HT-1080 cells were irradiated with 10 Gray and stained 2 h later with (a) anti-hSnm1 and (b) anti-hRad51; (c) merged fields after DAPI staining. HT-1080 cells were irradiated with 15 Gray and stained 9 h later with (d) anti-hSnm1 and (e) anti-hMre11; (f) merged fields after DAPI staining. Partial colocalization with BRCA1 ionizing radiation-induced foci was observed 5 h after 10 Gray, as shown by (g) anti-hSnm1 and (h) anti-BRCA1; (i) both fields merged with DAPI staining.

    Article Snippet: The Flag-hSnm1 open reading frame was subsequently cloned into the mammalian expression vector pEBS7 ( ) and also fused with enhanced green fluorescent protein (EGFP) with the pEGFP-C1 vector from Clontech.

    Techniques: Immunofluorescence, Irradiation, Staining

    hSnm1 is localized to the nucleus. Indirect immunofluorescence of MCF-7 cells probed with hSnm1 affinity-purified polyclonal antibodies displaying (a) diffuse nuclear staining and hSnm1 bodies (as indicated by white arrow) or (b) multiple nuclear foci. Epifluorescence of MCF-7 cells expressing EGFP-hSnm1 fusion protein showing localization to (c) a nuclear body and (d) foci (transfected cell indicated by white arrow). An undamaged HT-1080 cell transiently expressing the Flag-hSnm1 fusion construct was stained with (e) anti-hSnm1 antibodies and (f) anti-Flag M2 monoclonal antibodies. (g) The two images are shown merged after DAPI staining.

    Journal: Molecular and Cellular Biology

    Article Title: hSnm1 Colocalizes and Physically Associates with 53BP1 before and after DNA Damage

    doi: 10.1128/MCB.22.24.8635-8647.2002

    Figure Lengend Snippet: hSnm1 is localized to the nucleus. Indirect immunofluorescence of MCF-7 cells probed with hSnm1 affinity-purified polyclonal antibodies displaying (a) diffuse nuclear staining and hSnm1 bodies (as indicated by white arrow) or (b) multiple nuclear foci. Epifluorescence of MCF-7 cells expressing EGFP-hSnm1 fusion protein showing localization to (c) a nuclear body and (d) foci (transfected cell indicated by white arrow). An undamaged HT-1080 cell transiently expressing the Flag-hSnm1 fusion construct was stained with (e) anti-hSnm1 antibodies and (f) anti-Flag M2 monoclonal antibodies. (g) The two images are shown merged after DAPI staining.

    Article Snippet: The Flag-hSnm1 open reading frame was subsequently cloned into the mammalian expression vector pEBS7 ( ) and also fused with enhanced green fluorescent protein (EGFP) with the pEGFP-C1 vector from Clontech.

    Techniques: Immunofluorescence, Affinity Purification, Staining, Expressing, Transfection, Construct

    Colocalization of EGFP-hSnm1 protein with 53BP1. MCF-7 cells were transfected with pEGFP-hSnm1 followed by staining with anti-53BP1 and Toto-3. Localization of (a) EGFP-hSnm1 and (b) 53BP1 to a nuclear body in the same cell; (c) the two fields merged with Toto-3.

    Journal: Molecular and Cellular Biology

    Article Title: hSnm1 Colocalizes and Physically Associates with 53BP1 before and after DNA Damage

    doi: 10.1128/MCB.22.24.8635-8647.2002

    Figure Lengend Snippet: Colocalization of EGFP-hSnm1 protein with 53BP1. MCF-7 cells were transfected with pEGFP-hSnm1 followed by staining with anti-53BP1 and Toto-3. Localization of (a) EGFP-hSnm1 and (b) 53BP1 to a nuclear body in the same cell; (c) the two fields merged with Toto-3.

    Article Snippet: The Flag-hSnm1 open reading frame was subsequently cloned into the mammalian expression vector pEBS7 ( ) and also fused with enhanced green fluorescent protein (EGFP) with the pEGFP-C1 vector from Clontech.

    Techniques: Transfection, Staining

    Coimmunoprecipitation of 53BP1 and hSnm1. (A) HeLa cell extracts were incubated with beads only, preimmune serum, or affinity-purified anti-hSnm1 antibodies. Polyclonal anti-53BP1 antibodies were used to detect 53BP1 by immunoblotting. (B) HEK293 nuclear extracts were prepared with or without infection with EGFP-hSnm1-expressing adenovirus and immunoblotted with monoclonal antibodies against EGFP or polyclonal antibodies against 53BP1. Immunoprecipitations (IP) were performed with these extracts with anti-hSnm1 and anti-53BP1 antibodies as shown.

    Journal: Molecular and Cellular Biology

    Article Title: hSnm1 Colocalizes and Physically Associates with 53BP1 before and after DNA Damage

    doi: 10.1128/MCB.22.24.8635-8647.2002

    Figure Lengend Snippet: Coimmunoprecipitation of 53BP1 and hSnm1. (A) HeLa cell extracts were incubated with beads only, preimmune serum, or affinity-purified anti-hSnm1 antibodies. Polyclonal anti-53BP1 antibodies were used to detect 53BP1 by immunoblotting. (B) HEK293 nuclear extracts were prepared with or without infection with EGFP-hSnm1-expressing adenovirus and immunoblotted with monoclonal antibodies against EGFP or polyclonal antibodies against 53BP1. Immunoprecipitations (IP) were performed with these extracts with anti-hSnm1 and anti-53BP1 antibodies as shown.

    Article Snippet: The Flag-hSnm1 open reading frame was subsequently cloned into the mammalian expression vector pEBS7 ( ) and also fused with enhanced green fluorescent protein (EGFP) with the pEGFP-C1 vector from Clontech.

    Techniques: Incubation, Affinity Purification, Infection, Expressing

    Deletion analysis of hSNM1. (A) Various truncations and in-frame deletions were constructed in the hSNM1 segment of the EGFP-hSNM1 fusion gene. The solid black region indicates EGFP, and vertical lines indicate the conserved metallo-β-lactamase domain. Each construct was transfected into HT-1080 cells, and the location of the EGFP signal was determined 24 h later. (B) Expression of EGFP-hSnm1 deletion mutants in HT-1080 cells.

    Journal: Molecular and Cellular Biology

    Article Title: hSnm1 Colocalizes and Physically Associates with 53BP1 before and after DNA Damage

    doi: 10.1128/MCB.22.24.8635-8647.2002

    Figure Lengend Snippet: Deletion analysis of hSNM1. (A) Various truncations and in-frame deletions were constructed in the hSNM1 segment of the EGFP-hSNM1 fusion gene. The solid black region indicates EGFP, and vertical lines indicate the conserved metallo-β-lactamase domain. Each construct was transfected into HT-1080 cells, and the location of the EGFP signal was determined 24 h later. (B) Expression of EGFP-hSnm1 deletion mutants in HT-1080 cells.

    Article Snippet: The Flag-hSnm1 open reading frame was subsequently cloned into the mammalian expression vector pEBS7 ( ) and also fused with enhanced green fluorescent protein (EGFP) with the pEGFP-C1 vector from Clontech.

    Techniques: Construct, Transfection, Expressing

    (A) Northern blot of mRNAs from various tissues was probed with hSnm1 cDNA. A probe for β-actin was used to control for loading. (B) In vitro-translated Flag-hSnm1 protein labeled with [ 35 S]methionine was precipitated by anti-hSnm1-protein A-agarose beads, protein A beads only, or preimmune-protein A beads. B and SN indicate beads and supernatant, respectively.

    Journal: Molecular and Cellular Biology

    Article Title: hSnm1 Colocalizes and Physically Associates with 53BP1 before and after DNA Damage

    doi: 10.1128/MCB.22.24.8635-8647.2002

    Figure Lengend Snippet: (A) Northern blot of mRNAs from various tissues was probed with hSnm1 cDNA. A probe for β-actin was used to control for loading. (B) In vitro-translated Flag-hSnm1 protein labeled with [ 35 S]methionine was precipitated by anti-hSnm1-protein A-agarose beads, protein A beads only, or preimmune-protein A beads. B and SN indicate beads and supernatant, respectively.

    Article Snippet: The Flag-hSnm1 open reading frame was subsequently cloned into the mammalian expression vector pEBS7 ( ) and also fused with enhanced green fluorescent protein (EGFP) with the pEGFP-C1 vector from Clontech.

    Techniques: Northern Blot, In Vitro, Labeling

    Quantitative analysis of hSnm1 focus induction after DNA damage in MCF-7 cells by indirect immunofluorescence. hSnm1 antibodies were used to probe cells at various times after treatment with either ionizing radiation (IR) or 4HC. The percentages of cells displaying diffuse nuclear staining with fewer than 10 foci (open bars), hSnm1 bodies (hatched bars), and more than 10 foci (black bars) were calculated after scoring at least 100 nuclei for each time point. Reported here are the averages and standard deviations of three data sets.

    Journal: Molecular and Cellular Biology

    Article Title: hSnm1 Colocalizes and Physically Associates with 53BP1 before and after DNA Damage

    doi: 10.1128/MCB.22.24.8635-8647.2002

    Figure Lengend Snippet: Quantitative analysis of hSnm1 focus induction after DNA damage in MCF-7 cells by indirect immunofluorescence. hSnm1 antibodies were used to probe cells at various times after treatment with either ionizing radiation (IR) or 4HC. The percentages of cells displaying diffuse nuclear staining with fewer than 10 foci (open bars), hSnm1 bodies (hatched bars), and more than 10 foci (black bars) were calculated after scoring at least 100 nuclei for each time point. Reported here are the averages and standard deviations of three data sets.

    Article Snippet: The Flag-hSnm1 open reading frame was subsequently cloned into the mammalian expression vector pEBS7 ( ) and also fused with enhanced green fluorescent protein (EGFP) with the pEGFP-C1 vector from Clontech.

    Techniques: Immunofluorescence, Staining

    Quantitative analysis of hSnm1 nuclear staining during the cell cycle. (A) Histogram displaying the DNA content of asynchronous untreated MCF-7 cells analyzed by laser scanning cytometry and the observed frequencies of the different hSnm1 staining patterns corresponding to the G 1 , S, and G 2 subpopulations. PI, propidium iodide. (B) Similar data obtained from MCF-7 cells taken 5 h after treatment with 10 Gray of ionizing radiation. The percentages of cells displaying diffuse nuclear staining with fewer than 10 foci (white bars), hSnm1 bodies (hatched bars), and more than 10 foci (black bars) were calculated after scoring at least 150 nuclei for each time point.

    Journal: Molecular and Cellular Biology

    Article Title: hSnm1 Colocalizes and Physically Associates with 53BP1 before and after DNA Damage

    doi: 10.1128/MCB.22.24.8635-8647.2002

    Figure Lengend Snippet: Quantitative analysis of hSnm1 nuclear staining during the cell cycle. (A) Histogram displaying the DNA content of asynchronous untreated MCF-7 cells analyzed by laser scanning cytometry and the observed frequencies of the different hSnm1 staining patterns corresponding to the G 1 , S, and G 2 subpopulations. PI, propidium iodide. (B) Similar data obtained from MCF-7 cells taken 5 h after treatment with 10 Gray of ionizing radiation. The percentages of cells displaying diffuse nuclear staining with fewer than 10 foci (white bars), hSnm1 bodies (hatched bars), and more than 10 foci (black bars) were calculated after scoring at least 150 nuclei for each time point.

    Article Snippet: The Flag-hSnm1 open reading frame was subsequently cloned into the mammalian expression vector pEBS7 ( ) and also fused with enhanced green fluorescent protein (EGFP) with the pEGFP-C1 vector from Clontech.

    Techniques: Staining, Cytometry

    Colocalization of hSnm1 and 53BP1 in foci as detected by indirect immunofluorescence. MCF-7 cells were mock treated (a, b, and c) or treated with 10 Gray of ionizing radiation and fixed after 30 min (d, e, and f), after 90 min (g, h, and i), or after 5 h (j, k, and l). (a, d, g, and j) Polyclonal anti-hSnm1 staining with fluorescein isothiocyanate; (b, e, h, and k) monoclonal anti-53BP1 staining with tetramethyl rhodamine isocyanate; (c, f, i, and l) merged fields plus DAPI staining.

    Journal: Molecular and Cellular Biology

    Article Title: hSnm1 Colocalizes and Physically Associates with 53BP1 before and after DNA Damage

    doi: 10.1128/MCB.22.24.8635-8647.2002

    Figure Lengend Snippet: Colocalization of hSnm1 and 53BP1 in foci as detected by indirect immunofluorescence. MCF-7 cells were mock treated (a, b, and c) or treated with 10 Gray of ionizing radiation and fixed after 30 min (d, e, and f), after 90 min (g, h, and i), or after 5 h (j, k, and l). (a, d, g, and j) Polyclonal anti-hSnm1 staining with fluorescein isothiocyanate; (b, e, h, and k) monoclonal anti-53BP1 staining with tetramethyl rhodamine isocyanate; (c, f, i, and l) merged fields plus DAPI staining.

    Article Snippet: The Flag-hSnm1 open reading frame was subsequently cloned into the mammalian expression vector pEBS7 ( ) and also fused with enhanced green fluorescent protein (EGFP) with the pEGFP-C1 vector from Clontech.

    Techniques: Immunofluorescence, Staining

    The full-length cDNA sequence of Cydia pomonella CYP9A61 and deduced amino acid sequence. The start codon, stop codon, and polyadenylation signal sequences are underlined. The heme-binding domain and six substrate recognition sites (SRSs) are boxed. Dotted lines indicate the domains on which degenerate primers were designed.

    Journal: International Journal of Molecular Sciences

    Article Title: Molecular Cloning and Expression of CYP9A61: A Chlorpyrifos-Ethyl and Lambda-Cyhalothrin-Inducible Cytochrome P450 cDNA from Cydia pomonella

    doi: 10.3390/ijms141224211

    Figure Lengend Snippet: The full-length cDNA sequence of Cydia pomonella CYP9A61 and deduced amino acid sequence. The start codon, stop codon, and polyadenylation signal sequences are underlined. The heme-binding domain and six substrate recognition sites (SRSs) are boxed. Dotted lines indicate the domains on which degenerate primers were designed.

    Article Snippet: RNA Extraction and cDNA Synthesis for Molecular Cloning The total RNA of C. pomonella was extracted from five third instar larvae using the RNAiso Plus Kit based on the manufacturer’s instructions (Takara, Dalian, China).

    Techniques: Sequencing, Binding Assay

    Induction of C. pomonella CYP9A61 . RT-qPCR analysis relative expression of CYP9A61 treated with 12.5 ng chlorpyrifos-ethyl ( A ) and 0.19 ng lambda-cyhalothrin ( B ). Expression level was first normalized to the reference gene beta -actin . The normalized value of each sample was then divided by a specified control (the value of CYP9A61 in acetone treated control), and the ratio was applied to relative expression analysis. The results are shown as the mean ± SE. The error bars show the ranges of standard errors. Letters on the error bars indicate the significant differences by ANOVA analysis ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Molecular Cloning and Expression of CYP9A61: A Chlorpyrifos-Ethyl and Lambda-Cyhalothrin-Inducible Cytochrome P450 cDNA from Cydia pomonella

    doi: 10.3390/ijms141224211

    Figure Lengend Snippet: Induction of C. pomonella CYP9A61 . RT-qPCR analysis relative expression of CYP9A61 treated with 12.5 ng chlorpyrifos-ethyl ( A ) and 0.19 ng lambda-cyhalothrin ( B ). Expression level was first normalized to the reference gene beta -actin . The normalized value of each sample was then divided by a specified control (the value of CYP9A61 in acetone treated control), and the ratio was applied to relative expression analysis. The results are shown as the mean ± SE. The error bars show the ranges of standard errors. Letters on the error bars indicate the significant differences by ANOVA analysis ( p

    Article Snippet: RNA Extraction and cDNA Synthesis for Molecular Cloning The total RNA of C. pomonella was extracted from five third instar larvae using the RNAiso Plus Kit based on the manufacturer’s instructions (Takara, Dalian, China).

    Techniques: Quantitative RT-PCR, Expressing

    Phylogenetic relationship of C. pomonella CYP9A61 with 22 CYP9s from Lepidoptera. CYP9A61 (GenBank accession number: KC832920) is boxed. This un-rooted phylogenetic tree was constructed using the neighbor-joining method. Nodes indicate bootstrap calculated with 1000 replications support.

    Journal: International Journal of Molecular Sciences

    Article Title: Molecular Cloning and Expression of CYP9A61: A Chlorpyrifos-Ethyl and Lambda-Cyhalothrin-Inducible Cytochrome P450 cDNA from Cydia pomonella

    doi: 10.3390/ijms141224211

    Figure Lengend Snippet: Phylogenetic relationship of C. pomonella CYP9A61 with 22 CYP9s from Lepidoptera. CYP9A61 (GenBank accession number: KC832920) is boxed. This un-rooted phylogenetic tree was constructed using the neighbor-joining method. Nodes indicate bootstrap calculated with 1000 replications support.

    Article Snippet: RNA Extraction and cDNA Synthesis for Molecular Cloning The total RNA of C. pomonella was extracted from five third instar larvae using the RNAiso Plus Kit based on the manufacturer’s instructions (Takara, Dalian, China).

    Techniques: Construct

    Structure-based sequence alignments of Cydia pomonella CYP9A61, Manduca sexta CYP9A5, Heliothis virescens CYP9A1 and Bombyx mori CYP9A20 by ClustalW2. The predicted α-helices and β-sheets of CYP9A61 are marked on the top of sequences using boxes and arrows respectively. The β-domain is red, and α-domain is blue. The conserved regions of P450 enzymes including FxxGxxxCxG, PxxFxPxRF, ExxR, WxxxR are boxed using dashed lines.

    Journal: International Journal of Molecular Sciences

    Article Title: Molecular Cloning and Expression of CYP9A61: A Chlorpyrifos-Ethyl and Lambda-Cyhalothrin-Inducible Cytochrome P450 cDNA from Cydia pomonella

    doi: 10.3390/ijms141224211

    Figure Lengend Snippet: Structure-based sequence alignments of Cydia pomonella CYP9A61, Manduca sexta CYP9A5, Heliothis virescens CYP9A1 and Bombyx mori CYP9A20 by ClustalW2. The predicted α-helices and β-sheets of CYP9A61 are marked on the top of sequences using boxes and arrows respectively. The β-domain is red, and α-domain is blue. The conserved regions of P450 enzymes including FxxGxxxCxG, PxxFxPxRF, ExxR, WxxxR are boxed using dashed lines.

    Article Snippet: RNA Extraction and cDNA Synthesis for Molecular Cloning The total RNA of C. pomonella was extracted from five third instar larvae using the RNAiso Plus Kit based on the manufacturer’s instructions (Takara, Dalian, China).

    Techniques: Sequencing

    Comparison of C. pomonella CYP9A61 and CYP9A12 (EU541248.2) and CYP9A17 (EU541247.1) from Helicoverpa armigera . SRS1, SRS4 and SRS5 are boxed. Asterisks ( * ) indicate identical residues among three sequences; colons (:) indicate residues with conserved substitutions; dots (.) represent residues with weakly conserved residues in the three sequence alignment.

    Journal: International Journal of Molecular Sciences

    Article Title: Molecular Cloning and Expression of CYP9A61: A Chlorpyrifos-Ethyl and Lambda-Cyhalothrin-Inducible Cytochrome P450 cDNA from Cydia pomonella

    doi: 10.3390/ijms141224211

    Figure Lengend Snippet: Comparison of C. pomonella CYP9A61 and CYP9A12 (EU541248.2) and CYP9A17 (EU541247.1) from Helicoverpa armigera . SRS1, SRS4 and SRS5 are boxed. Asterisks ( * ) indicate identical residues among three sequences; colons (:) indicate residues with conserved substitutions; dots (.) represent residues with weakly conserved residues in the three sequence alignment.

    Article Snippet: RNA Extraction and cDNA Synthesis for Molecular Cloning The total RNA of C. pomonella was extracted from five third instar larvae using the RNAiso Plus Kit based on the manufacturer’s instructions (Takara, Dalian, China).

    Techniques: Sequencing

    MFO activities of C. pomonella treated with 2.5 ng chlorpyrifos-ethyl and 0.19 ng lambda-cyhalothrin using PNOD as substrate. The results are expressed as pmole p -nitrophenol min −1 mg of protein −1 . The error bars represent the standard error across three replicates. Asterisks on the error bars indicate the significant differences between chlorpyrifos-ethyl or lambda-cyhalothrin treatment and control by Student’s t -test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

    Journal: International Journal of Molecular Sciences

    Article Title: Molecular Cloning and Expression of CYP9A61: A Chlorpyrifos-Ethyl and Lambda-Cyhalothrin-Inducible Cytochrome P450 cDNA from Cydia pomonella

    doi: 10.3390/ijms141224211

    Figure Lengend Snippet: MFO activities of C. pomonella treated with 2.5 ng chlorpyrifos-ethyl and 0.19 ng lambda-cyhalothrin using PNOD as substrate. The results are expressed as pmole p -nitrophenol min −1 mg of protein −1 . The error bars represent the standard error across three replicates. Asterisks on the error bars indicate the significant differences between chlorpyrifos-ethyl or lambda-cyhalothrin treatment and control by Student’s t -test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

    Article Snippet: RNA Extraction and cDNA Synthesis for Molecular Cloning The total RNA of C. pomonella was extracted from five third instar larvae using the RNAiso Plus Kit based on the manufacturer’s instructions (Takara, Dalian, China).

    Techniques:

    Relative expression level of C. pomonella CYP9A61 in different developmental stages ( A ) and tissues ( B ). Expression level was normalized using β-actin as the standard. The normalized value was applied to relative expression analysis. The results are shown as the mean ± SE. The error bars show the ranges of standard errors. Letters or asterisks on the error bars indicate the significant differences by ANOVA analysis ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Molecular Cloning and Expression of CYP9A61: A Chlorpyrifos-Ethyl and Lambda-Cyhalothrin-Inducible Cytochrome P450 cDNA from Cydia pomonella

    doi: 10.3390/ijms141224211

    Figure Lengend Snippet: Relative expression level of C. pomonella CYP9A61 in different developmental stages ( A ) and tissues ( B ). Expression level was normalized using β-actin as the standard. The normalized value was applied to relative expression analysis. The results are shown as the mean ± SE. The error bars show the ranges of standard errors. Letters or asterisks on the error bars indicate the significant differences by ANOVA analysis ( p

    Article Snippet: RNA Extraction and cDNA Synthesis for Molecular Cloning The total RNA of C. pomonella was extracted from five third instar larvae using the RNAiso Plus Kit based on the manufacturer’s instructions (Takara, Dalian, China).

    Techniques: Expressing

    IRES activity of the ATXN8OS transcript. (A) ATXN8OS organization with promoter (open arrow), exons (open boxes) and functional splice donor sequences (GT) of D exons (D5, D4, D, D″ and D′) indicated. The CTG repeat tract is located in exon A. Transcription start site of exon D5 and exon D are represented by +1 and +801, respectively. (B) ATXN8OS RNA (NR_002717) generated from the splicing events represented by the wavy lines. The putative ORF initiated from AUG +1247 is indicated by the open boxes inside the RNA. The restriction enzymes and the cutting sites used to generate +801∼+1195 cDNA fragment of ATXN8OS are shown on the bottom of the cDNA. (C) The dual luciferase reporter plasmid had Renilla luciferase and firefly luciferase genes between the TK promoter and polyadenylation signal. The locations of Xba I, Xho I and Bam HI sites used for construction are shown on the top. (D) Relative luciferase activities generated by dual luciferase constructs with ECMV IRES and ATXN8OS +801∼+1195 cDNA fragment in HEK-293 and IMR-32 cells. Forty-eight hours following transfection, cells were harvested and luciferases activities were measured. IRES activity is expressed as percentages of the activity of the ECMV IRES, which was set at 100%. In addition, relative luciferase activities with ATXN8OS +801∼+953 and +953∼+1195 cDNA fragments were measured in HEK-293 cells, with IRES activity of +801∼+1195 set at 100%. Each value is the mean ± SD of three independent experiments each performed in duplicate.

    Journal: PLoS ONE

    Article Title: Internal Ribosome Entry Segment Activity of ATXN8 Opposite Strand RNA

    doi: 10.1371/journal.pone.0073885

    Figure Lengend Snippet: IRES activity of the ATXN8OS transcript. (A) ATXN8OS organization with promoter (open arrow), exons (open boxes) and functional splice donor sequences (GT) of D exons (D5, D4, D, D″ and D′) indicated. The CTG repeat tract is located in exon A. Transcription start site of exon D5 and exon D are represented by +1 and +801, respectively. (B) ATXN8OS RNA (NR_002717) generated from the splicing events represented by the wavy lines. The putative ORF initiated from AUG +1247 is indicated by the open boxes inside the RNA. The restriction enzymes and the cutting sites used to generate +801∼+1195 cDNA fragment of ATXN8OS are shown on the bottom of the cDNA. (C) The dual luciferase reporter plasmid had Renilla luciferase and firefly luciferase genes between the TK promoter and polyadenylation signal. The locations of Xba I, Xho I and Bam HI sites used for construction are shown on the top. (D) Relative luciferase activities generated by dual luciferase constructs with ECMV IRES and ATXN8OS +801∼+1195 cDNA fragment in HEK-293 and IMR-32 cells. Forty-eight hours following transfection, cells were harvested and luciferases activities were measured. IRES activity is expressed as percentages of the activity of the ECMV IRES, which was set at 100%. In addition, relative luciferase activities with ATXN8OS +801∼+953 and +953∼+1195 cDNA fragments were measured in HEK-293 cells, with IRES activity of +801∼+1195 set at 100%. Each value is the mean ± SD of three independent experiments each performed in duplicate.

    Article Snippet: The ATXN8OS cDNA were then cloned into the Eco RI site of pEGFP-N1 (Clontech).

    Techniques: Activity Assay, Functional Assay, CTG Assay, Generated, Luciferase, Plasmid Preparation, Construct, Transfection

    Transient expression of ATXN8OS ORF-EGFP constructs in HEK-293 cells. (A) ORF-EGFP constructs. A 752-bp cDNA fragment containing exon D, C2 and portion of C1 was inserted into pEGFP-N1 MCS so that ATXN8OS ORF was fused in-frame with the EGFP gene to generate pCMV/+801. A +1∼+800 ATXN8OS fragment was inserted between CMV promoter and exon D of pCMV/+801 to generate pCMV/+1. In pATXN8OS/−114 and/−481, 114 and 481-bp ATXN8OS promoter fragments was used to replace the CMV promoter in pCMV/+1. (B) Real-time PCR quantification of ORF-EGFP RNA level relative to endogenous HPRT1 RNA. To normalize, expression level in pATXN8OS/−481 transfected cells is set as 1.0. (C) FACS analysis of EGFP fluorescence. Levels of EGFP were expressed as percentages of pIRES2-EGFP, which was set at 100%. Each value is the mean ± SD of three independent experiments each performed in duplicate.

    Journal: PLoS ONE

    Article Title: Internal Ribosome Entry Segment Activity of ATXN8 Opposite Strand RNA

    doi: 10.1371/journal.pone.0073885

    Figure Lengend Snippet: Transient expression of ATXN8OS ORF-EGFP constructs in HEK-293 cells. (A) ORF-EGFP constructs. A 752-bp cDNA fragment containing exon D, C2 and portion of C1 was inserted into pEGFP-N1 MCS so that ATXN8OS ORF was fused in-frame with the EGFP gene to generate pCMV/+801. A +1∼+800 ATXN8OS fragment was inserted between CMV promoter and exon D of pCMV/+801 to generate pCMV/+1. In pATXN8OS/−114 and/−481, 114 and 481-bp ATXN8OS promoter fragments was used to replace the CMV promoter in pCMV/+1. (B) Real-time PCR quantification of ORF-EGFP RNA level relative to endogenous HPRT1 RNA. To normalize, expression level in pATXN8OS/−481 transfected cells is set as 1.0. (C) FACS analysis of EGFP fluorescence. Levels of EGFP were expressed as percentages of pIRES2-EGFP, which was set at 100%. Each value is the mean ± SD of three independent experiments each performed in duplicate.

    Article Snippet: The ATXN8OS cDNA were then cloned into the Eco RI site of pEGFP-N1 (Clontech).

    Techniques: Expressing, Construct, Real-time Polymerase Chain Reaction, Transfection, FACS, Fluorescence