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  • 99
    Thermo Fisher lipofectamine rnaimax
    Lipofectamine Rnaimax, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 50858 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology carm1
    <t>CARM1</t> is associated with active HTLV-1 LTR chromatin in vivo. SP cells, which contain a single active integrated copy of the HTLV-1 proviral genome, were subjected to ChIP assays. Antibodies specific for Tax, CARM1, dimethyl histone H3 (R2, R17, R26, or K9), histone H3, and acetylated histone H3 (K9) were used for immunoprecipitation. PCRs were carried out to analyze precipitated DNA using primers specific for the HTLV-1 LTR and β-globin promoter region.
    Carm1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher pcdna3
    CTAR2 is the major site of LMP1-mediated Fascin induction. (A) Scheme of LMP1 and its signaling domains, the C-terminal activating regions 1 (CTAR1; aa 194–231) and 2 (CTAR2; aa 351–386). Mutations of the P(204)xQxT consensus motif to AxAxA in CTAR1 and deletion of aa 371–386 in CTAR2 are indicated. Signaling pathways induced by LMP1 are shown in rectangular boxes, adaptor molecules in rounded boxes. can. indicates canonical; noncan., noncanonical. (B) qPCR of Fascin transcripts 48 h after transfection of Jurkat cells with wt-LMP1 (20 μg pCMV-HA-LMP1), LMP1 mutants (40 μg pCMV-HA-LMP1(AAA), 20 μg of pCMV-HA-LMP1-Δ371-386) and HTLV-1/Tax (40 μg pcTax-1) in comparison to mock-transfected cells (100 μg <t>pcDNA3).</t> Total transfected DNA was adjusted to 100 μg with pcDNA3. Copy numbers were normalized to those of ACTB and on mock-transfected cells. Mean values +/− standard error (SE) are given. (C) Detection of Fascin and LMP1 by immunoblot after transient transfection of Jurkat cells with the constructs described in (B) . Fascin expression was quantitatively evaluated by densitometry and normalized on LMP1 protein expression. The mean of three independent experiments +/−SE is shown. **indicates P
    Pcdna3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 45292 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega renilla luciferase reporter plasmid
    CTAR2 is the major site of LMP1-mediated Fascin induction. (A) Scheme of LMP1 and its signaling domains, the C-terminal activating regions 1 (CTAR1; aa 194–231) and 2 (CTAR2; aa 351–386). Mutations of the P(204)xQxT consensus motif to AxAxA in CTAR1 and deletion of aa 371–386 in CTAR2 are indicated. Signaling pathways induced by LMP1 are shown in rectangular boxes, adaptor molecules in rounded boxes. can. indicates canonical; noncan., noncanonical. (B) qPCR of Fascin transcripts 48 h after transfection of Jurkat cells with wt-LMP1 (20 μg pCMV-HA-LMP1), LMP1 mutants (40 μg pCMV-HA-LMP1(AAA), 20 μg of pCMV-HA-LMP1-Δ371-386) and HTLV-1/Tax (40 μg pcTax-1) in comparison to mock-transfected cells (100 μg <t>pcDNA3).</t> Total transfected DNA was adjusted to 100 μg with pcDNA3. Copy numbers were normalized to those of ACTB and on mock-transfected cells. Mean values +/− standard error (SE) are given. (C) Detection of Fascin and LMP1 by immunoblot after transient transfection of Jurkat cells with the constructs described in (B) . Fascin expression was quantitatively evaluated by densitometry and normalized on LMP1 protein expression. The mean of three independent experiments +/−SE is shown. **indicates P
    Renilla Luciferase Reporter Plasmid, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 924 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad gene pulser x electroporation system
    CTAR2 is the major site of LMP1-mediated Fascin induction. (A) Scheme of LMP1 and its signaling domains, the C-terminal activating regions 1 (CTAR1; aa 194–231) and 2 (CTAR2; aa 351–386). Mutations of the P(204)xQxT consensus motif to AxAxA in CTAR1 and deletion of aa 371–386 in CTAR2 are indicated. Signaling pathways induced by LMP1 are shown in rectangular boxes, adaptor molecules in rounded boxes. can. indicates canonical; noncan., noncanonical. (B) qPCR of Fascin transcripts 48 h after transfection of Jurkat cells with wt-LMP1 (20 μg pCMV-HA-LMP1), LMP1 mutants (40 μg pCMV-HA-LMP1(AAA), 20 μg of pCMV-HA-LMP1-Δ371-386) and HTLV-1/Tax (40 μg pcTax-1) in comparison to mock-transfected cells (100 μg <t>pcDNA3).</t> Total transfected DNA was adjusted to 100 μg with pcDNA3. Copy numbers were normalized to those of ACTB and on mock-transfected cells. Mean values +/− standard error (SE) are given. (C) Detection of Fascin and LMP1 by immunoblot after transient transfection of Jurkat cells with the constructs described in (B) . Fascin expression was quantitatively evaluated by densitometry and normalized on LMP1 protein expression. The mean of three independent experiments +/−SE is shown. **indicates P
    Gene Pulser X Electroporation System, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher plasmid pcdna6
    CTAR2 is the major site of LMP1-mediated Fascin induction. (A) Scheme of LMP1 and its signaling domains, the C-terminal activating regions 1 (CTAR1; aa 194–231) and 2 (CTAR2; aa 351–386). Mutations of the P(204)xQxT consensus motif to AxAxA in CTAR1 and deletion of aa 371–386 in CTAR2 are indicated. Signaling pathways induced by LMP1 are shown in rectangular boxes, adaptor molecules in rounded boxes. can. indicates canonical; noncan., noncanonical. (B) qPCR of Fascin transcripts 48 h after transfection of Jurkat cells with wt-LMP1 (20 μg pCMV-HA-LMP1), LMP1 mutants (40 μg pCMV-HA-LMP1(AAA), 20 μg of pCMV-HA-LMP1-Δ371-386) and HTLV-1/Tax (40 μg pcTax-1) in comparison to mock-transfected cells (100 μg <t>pcDNA3).</t> Total transfected DNA was adjusted to 100 μg with pcDNA3. Copy numbers were normalized to those of ACTB and on mock-transfected cells. Mean values +/− standard error (SE) are given. (C) Detection of Fascin and LMP1 by immunoblot after transient transfection of Jurkat cells with the constructs described in (B) . Fascin expression was quantitatively evaluated by densitometry and normalized on LMP1 protein expression. The mean of three independent experiments +/−SE is shown. **indicates P
    Plasmid Pcdna6, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    ZeptoMetrix transfection supernatants
    CTAR2 is the major site of LMP1-mediated Fascin induction. (A) Scheme of LMP1 and its signaling domains, the C-terminal activating regions 1 (CTAR1; aa 194–231) and 2 (CTAR2; aa 351–386). Mutations of the P(204)xQxT consensus motif to AxAxA in CTAR1 and deletion of aa 371–386 in CTAR2 are indicated. Signaling pathways induced by LMP1 are shown in rectangular boxes, adaptor molecules in rounded boxes. can. indicates canonical; noncan., noncanonical. (B) qPCR of Fascin transcripts 48 h after transfection of Jurkat cells with wt-LMP1 (20 μg pCMV-HA-LMP1), LMP1 mutants (40 μg pCMV-HA-LMP1(AAA), 20 μg of pCMV-HA-LMP1-Δ371-386) and HTLV-1/Tax (40 μg pcTax-1) in comparison to mock-transfected cells (100 μg <t>pcDNA3).</t> Total transfected DNA was adjusted to 100 μg with pcDNA3. Copy numbers were normalized to those of ACTB and on mock-transfected cells. Mean values +/− standard error (SE) are given. (C) Detection of Fascin and LMP1 by immunoblot after transient transfection of Jurkat cells with the constructs described in (B) . Fascin expression was quantitatively evaluated by densitometry and normalized on LMP1 protein expression. The mean of three independent experiments +/−SE is shown. **indicates P
    Transfection Supernatants, supplied by ZeptoMetrix, used in various techniques. Bioz Stars score: 88/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher transfections
    CTAR2 is the major site of LMP1-mediated Fascin induction. (A) Scheme of LMP1 and its signaling domains, the C-terminal activating regions 1 (CTAR1; aa 194–231) and 2 (CTAR2; aa 351–386). Mutations of the P(204)xQxT consensus motif to AxAxA in CTAR1 and deletion of aa 371–386 in CTAR2 are indicated. Signaling pathways induced by LMP1 are shown in rectangular boxes, adaptor molecules in rounded boxes. can. indicates canonical; noncan., noncanonical. (B) qPCR of Fascin transcripts 48 h after transfection of Jurkat cells with wt-LMP1 (20 μg pCMV-HA-LMP1), LMP1 mutants (40 μg pCMV-HA-LMP1(AAA), 20 μg of pCMV-HA-LMP1-Δ371-386) and HTLV-1/Tax (40 μg pcTax-1) in comparison to mock-transfected cells (100 μg <t>pcDNA3).</t> Total transfected DNA was adjusted to 100 μg with pcDNA3. Copy numbers were normalized to those of ACTB and on mock-transfected cells. Mean values +/− standard error (SE) are given. (C) Detection of Fascin and LMP1 by immunoblot after transient transfection of Jurkat cells with the constructs described in (B) . Fascin expression was quantitatively evaluated by densitometry and normalized on LMP1 protein expression. The mean of three independent experiments +/−SE is shown. **indicates P
    Transfections, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9824 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    ZeptoMetrix htlv 1 p19 gag elisa
    CTAR2 is the major site of LMP1-mediated Fascin induction. (A) Scheme of LMP1 and its signaling domains, the C-terminal activating regions 1 (CTAR1; aa 194–231) and 2 (CTAR2; aa 351–386). Mutations of the P(204)xQxT consensus motif to AxAxA in CTAR1 and deletion of aa 371–386 in CTAR2 are indicated. Signaling pathways induced by LMP1 are shown in rectangular boxes, adaptor molecules in rounded boxes. can. indicates canonical; noncan., noncanonical. (B) qPCR of Fascin transcripts 48 h after transfection of Jurkat cells with wt-LMP1 (20 μg pCMV-HA-LMP1), LMP1 mutants (40 μg pCMV-HA-LMP1(AAA), 20 μg of pCMV-HA-LMP1-Δ371-386) and HTLV-1/Tax (40 μg pcTax-1) in comparison to mock-transfected cells (100 μg <t>pcDNA3).</t> Total transfected DNA was adjusted to 100 μg with pcDNA3. Copy numbers were normalized to those of ACTB and on mock-transfected cells. Mean values +/− standard error (SE) are given. (C) Detection of Fascin and LMP1 by immunoblot after transient transfection of Jurkat cells with the constructs described in (B) . Fascin expression was quantitatively evaluated by densitometry and normalized on LMP1 protein expression. The mean of three independent experiments +/−SE is shown. **indicates P
    Htlv 1 P19 Gag Elisa, supplied by ZeptoMetrix, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pasteur Institute hiv 1 ff luc vector
    SAMHD1 suppresses gene expression driven by the LTR from <t>HIV-1</t> or HTLV-1 but not from MLV. (A to F) HEK293T cells were transfected with an empty vector (V) or increasing amounts of constructs expressing HA-tagged SAMHD1 and either an HIV-1 LTR-driven <t>FF-Luc</t> construct with (+) or without (−) an HIV-1 Tat-expressing plasmid (A and B), an HTLV-1 LTR-driven FF-Luc construct with or without an HTLV-1 Tax-encoding plasmid (C and D), or an MLV LTR-driven FF-Luc construct (E and F). Overexpression of SAMHD1 was analyzed by immunoblotting (A, C, and E) with GAPDH as a loading control. Cotransfection of Ren-Luc was used as a control of transfection efficiency, with LTR-driven FF-Luc activity normalized to Ren-Luc activity. (B, D, and F) Luciferase activity was determined 24 h posttransfection. Error bars show standard errors of the means for three (HIV-Luc with or without Tat, HTLV-Luc with Tax) or two (HTLV-Luc without Tax, MLV-Luc) independent experiments. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple-comparison posttest. Asterisks indicate significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001) from results for vector (V) control cells.
    Hiv 1 Ff Luc Vector, supplied by Pasteur Institute, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Bio-Rad electroporation
    SAMHD1 suppresses gene expression driven by the LTR from <t>HIV-1</t> or HTLV-1 but not from MLV. (A to F) HEK293T cells were transfected with an empty vector (V) or increasing amounts of constructs expressing HA-tagged SAMHD1 and either an HIV-1 LTR-driven <t>FF-Luc</t> construct with (+) or without (−) an HIV-1 Tat-expressing plasmid (A and B), an HTLV-1 LTR-driven FF-Luc construct with or without an HTLV-1 Tax-encoding plasmid (C and D), or an MLV LTR-driven FF-Luc construct (E and F). Overexpression of SAMHD1 was analyzed by immunoblotting (A, C, and E) with GAPDH as a loading control. Cotransfection of Ren-Luc was used as a control of transfection efficiency, with LTR-driven FF-Luc activity normalized to Ren-Luc activity. (B, D, and F) Luciferase activity was determined 24 h posttransfection. Error bars show standard errors of the means for three (HIV-Luc with or without Tat, HTLV-Luc with Tax) or two (HTLV-Luc without Tax, MLV-Luc) independent experiments. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple-comparison posttest. Asterisks indicate significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001) from results for vector (V) control cells.
    Electroporation, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 14452 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega dual luciferase kit
    SAMHD1 suppresses gene expression driven by the LTR from <t>HIV-1</t> or HTLV-1 but not from MLV. (A to F) HEK293T cells were transfected with an empty vector (V) or increasing amounts of constructs expressing HA-tagged SAMHD1 and either an HIV-1 LTR-driven <t>FF-Luc</t> construct with (+) or without (−) an HIV-1 Tat-expressing plasmid (A and B), an HTLV-1 LTR-driven FF-Luc construct with or without an HTLV-1 Tax-encoding plasmid (C and D), or an MLV LTR-driven FF-Luc construct (E and F). Overexpression of SAMHD1 was analyzed by immunoblotting (A, C, and E) with GAPDH as a loading control. Cotransfection of Ren-Luc was used as a control of transfection efficiency, with LTR-driven FF-Luc activity normalized to Ren-Luc activity. (B, D, and F) Luciferase activity was determined 24 h posttransfection. Error bars show standard errors of the means for three (HIV-Luc with or without Tat, HTLV-Luc with Tax) or two (HTLV-Luc without Tax, MLV-Luc) independent experiments. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple-comparison posttest. Asterisks indicate significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001) from results for vector (V) control cells.
    Dual Luciferase Kit, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 6249 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen lipid reagent
    SAMHD1 suppresses gene expression driven by the LTR from <t>HIV-1</t> or HTLV-1 but not from MLV. (A to F) HEK293T cells were transfected with an empty vector (V) or increasing amounts of constructs expressing HA-tagged SAMHD1 and either an HIV-1 LTR-driven <t>FF-Luc</t> construct with (+) or without (−) an HIV-1 Tat-expressing plasmid (A and B), an HTLV-1 LTR-driven FF-Luc construct with or without an HTLV-1 Tax-encoding plasmid (C and D), or an MLV LTR-driven FF-Luc construct (E and F). Overexpression of SAMHD1 was analyzed by immunoblotting (A, C, and E) with GAPDH as a loading control. Cotransfection of Ren-Luc was used as a control of transfection efficiency, with LTR-driven FF-Luc activity normalized to Ren-Luc activity. (B, D, and F) Luciferase activity was determined 24 h posttransfection. Error bars show standard errors of the means for three (HIV-Luc with or without Tat, HTLV-Luc with Tax) or two (HTLV-Luc without Tax, MLV-Luc) independent experiments. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple-comparison posttest. Asterisks indicate significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001) from results for vector (V) control cells.
    Lipid Reagent, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Stratagene pbc sk
    SAMHD1 suppresses gene expression driven by the LTR from <t>HIV-1</t> or HTLV-1 but not from MLV. (A to F) HEK293T cells were transfected with an empty vector (V) or increasing amounts of constructs expressing HA-tagged SAMHD1 and either an HIV-1 LTR-driven <t>FF-Luc</t> construct with (+) or without (−) an HIV-1 Tat-expressing plasmid (A and B), an HTLV-1 LTR-driven FF-Luc construct with or without an HTLV-1 Tax-encoding plasmid (C and D), or an MLV LTR-driven FF-Luc construct (E and F). Overexpression of SAMHD1 was analyzed by immunoblotting (A, C, and E) with GAPDH as a loading control. Cotransfection of Ren-Luc was used as a control of transfection efficiency, with LTR-driven FF-Luc activity normalized to Ren-Luc activity. (B, D, and F) Luciferase activity was determined 24 h posttransfection. Error bars show standard errors of the means for three (HIV-Luc with or without Tat, HTLV-Luc with Tax) or two (HTLV-Luc without Tax, MLV-Luc) independent experiments. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple-comparison posttest. Asterisks indicate significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001) from results for vector (V) control cells.
    Pbc Sk, supplied by Stratagene, used in various techniques. Bioz Stars score: 91/100, based on 617 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Agilent technologies quikchange mutagenesis kit
    SAMHD1 suppresses gene expression driven by the LTR from <t>HIV-1</t> or HTLV-1 but not from MLV. (A to F) HEK293T cells were transfected with an empty vector (V) or increasing amounts of constructs expressing HA-tagged SAMHD1 and either an HIV-1 LTR-driven <t>FF-Luc</t> construct with (+) or without (−) an HIV-1 Tat-expressing plasmid (A and B), an HTLV-1 LTR-driven FF-Luc construct with or without an HTLV-1 Tax-encoding plasmid (C and D), or an MLV LTR-driven FF-Luc construct (E and F). Overexpression of SAMHD1 was analyzed by immunoblotting (A, C, and E) with GAPDH as a loading control. Cotransfection of Ren-Luc was used as a control of transfection efficiency, with LTR-driven FF-Luc activity normalized to Ren-Luc activity. (B, D, and F) Luciferase activity was determined 24 h posttransfection. Error bars show standard errors of the means for three (HIV-Luc with or without Tat, HTLV-Luc with Tax) or two (HTLV-Luc without Tax, MLV-Luc) independent experiments. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple-comparison posttest. Asterisks indicate significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001) from results for vector (V) control cells.
    Quikchange Mutagenesis Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 3075 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Inserm Transfert rxre cat
    SAMHD1 suppresses gene expression driven by the LTR from <t>HIV-1</t> or HTLV-1 but not from MLV. (A to F) HEK293T cells were transfected with an empty vector (V) or increasing amounts of constructs expressing HA-tagged SAMHD1 and either an HIV-1 LTR-driven <t>FF-Luc</t> construct with (+) or without (−) an HIV-1 Tat-expressing plasmid (A and B), an HTLV-1 LTR-driven FF-Luc construct with or without an HTLV-1 Tax-encoding plasmid (C and D), or an MLV LTR-driven FF-Luc construct (E and F). Overexpression of SAMHD1 was analyzed by immunoblotting (A, C, and E) with GAPDH as a loading control. Cotransfection of Ren-Luc was used as a control of transfection efficiency, with LTR-driven FF-Luc activity normalized to Ren-Luc activity. (B, D, and F) Luciferase activity was determined 24 h posttransfection. Error bars show standard errors of the means for three (HIV-Luc with or without Tat, HTLV-Luc with Tax) or two (HTLV-Luc without Tax, MLV-Luc) independent experiments. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple-comparison posttest. Asterisks indicate significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001) from results for vector (V) control cells.
    Rxre Cat, supplied by Inserm Transfert, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen superfect reagent
    SAMHD1 suppresses gene expression driven by the LTR from <t>HIV-1</t> or HTLV-1 but not from MLV. (A to F) HEK293T cells were transfected with an empty vector (V) or increasing amounts of constructs expressing HA-tagged SAMHD1 and either an HIV-1 LTR-driven <t>FF-Luc</t> construct with (+) or without (−) an HIV-1 Tat-expressing plasmid (A and B), an HTLV-1 LTR-driven FF-Luc construct with or without an HTLV-1 Tax-encoding plasmid (C and D), or an MLV LTR-driven FF-Luc construct (E and F). Overexpression of SAMHD1 was analyzed by immunoblotting (A, C, and E) with GAPDH as a loading control. Cotransfection of Ren-Luc was used as a control of transfection efficiency, with LTR-driven FF-Luc activity normalized to Ren-Luc activity. (B, D, and F) Luciferase activity was determined 24 h posttransfection. Error bars show standard errors of the means for three (HIV-Luc with or without Tat, HTLV-Luc with Tax) or two (HTLV-Luc without Tax, MLV-Luc) independent experiments. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple-comparison posttest. Asterisks indicate significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001) from results for vector (V) control cells.
    Superfect Reagent, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 5582 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    CARM1 is associated with active HTLV-1 LTR chromatin in vivo. SP cells, which contain a single active integrated copy of the HTLV-1 proviral genome, were subjected to ChIP assays. Antibodies specific for Tax, CARM1, dimethyl histone H3 (R2, R17, R26, or K9), histone H3, and acetylated histone H3 (K9) were used for immunoprecipitation. PCRs were carried out to analyze precipitated DNA using primers specific for the HTLV-1 LTR and β-globin promoter region.

    Journal: Journal of Virology

    Article Title: Coactivator-Associated Arginine Methyltransferase 1 Enhances Transcriptional Activity of the Human T-Cell Lymphotropic Virus Type 1 Long Terminal Repeat through Direct Interaction with Tax

    doi: 10.1128/JVI.00186-06

    Figure Lengend Snippet: CARM1 is associated with active HTLV-1 LTR chromatin in vivo. SP cells, which contain a single active integrated copy of the HTLV-1 proviral genome, were subjected to ChIP assays. Antibodies specific for Tax, CARM1, dimethyl histone H3 (R2, R17, R26, or K9), histone H3, and acetylated histone H3 (K9) were used for immunoprecipitation. PCRs were carried out to analyze precipitated DNA using primers specific for the HTLV-1 LTR and β-globin promoter region.

    Article Snippet: To examine whether CARM1 plays a role in Tax transactivation of the HTLV-1 LTR, we first compared the relative level of Tax transactivation in the presence and absence of exogenous CARM1.

    Techniques: In Vivo, Chromatin Immunoprecipitation, Immunoprecipitation

    CARM1 is recruited to the HTLV-1 PICs in the presence of Tax. HTLV-1 PICs were assembled by incubating biotinylated HTLV-1 templates with HeLa nuclear extracts (ext) in the absence or presence of the His 6 -Tax WT or mutant (del 151-204) and then purified with streptavidin-coated magnetic beads. The protein components of the purified PICs were analyzed by Western blotting with anti-Tax (A), -CARM1 (B), -CREB (C), or -p300 (D) antibodies. DNA-bio, biotinylated DNA.

    Journal: Journal of Virology

    Article Title: Coactivator-Associated Arginine Methyltransferase 1 Enhances Transcriptional Activity of the Human T-Cell Lymphotropic Virus Type 1 Long Terminal Repeat through Direct Interaction with Tax

    doi: 10.1128/JVI.00186-06

    Figure Lengend Snippet: CARM1 is recruited to the HTLV-1 PICs in the presence of Tax. HTLV-1 PICs were assembled by incubating biotinylated HTLV-1 templates with HeLa nuclear extracts (ext) in the absence or presence of the His 6 -Tax WT or mutant (del 151-204) and then purified with streptavidin-coated magnetic beads. The protein components of the purified PICs were analyzed by Western blotting with anti-Tax (A), -CARM1 (B), -CREB (C), or -p300 (D) antibodies. DNA-bio, biotinylated DNA.

    Article Snippet: To examine whether CARM1 plays a role in Tax transactivation of the HTLV-1 LTR, we first compared the relative level of Tax transactivation in the presence and absence of exogenous CARM1.

    Techniques: Mutagenesis, Purification, Magnetic Beads, Western Blot

    CARM1 mutant does not influence Tax transcription of the HTLV-1 LTR. (A) HeLa cells were transiently transfected using Effectene transfection reagent (QIAGEN) with reporter construct HTLV-1 LTR-Luc (0.1 μg), RSV β-Gal (0.05 μg), CARM1 WT or MT (0.1 μg), or Tax (0.1 μg) expression plasmids. At 24 h posttransfection, cells were collected and luciferase activities were measured. Luciferase values were adjusted for transfection efficiency using RSV β-Gal. The graph represents the luciferase activity from three independent experiments. The standard deviation for the three experiments is included. (B) Western blot analysis was performed for Tax (Tab172) and CARM1 (Upstate).

    Journal: Journal of Virology

    Article Title: Coactivator-Associated Arginine Methyltransferase 1 Enhances Transcriptional Activity of the Human T-Cell Lymphotropic Virus Type 1 Long Terminal Repeat through Direct Interaction with Tax

    doi: 10.1128/JVI.00186-06

    Figure Lengend Snippet: CARM1 mutant does not influence Tax transcription of the HTLV-1 LTR. (A) HeLa cells were transiently transfected using Effectene transfection reagent (QIAGEN) with reporter construct HTLV-1 LTR-Luc (0.1 μg), RSV β-Gal (0.05 μg), CARM1 WT or MT (0.1 μg), or Tax (0.1 μg) expression plasmids. At 24 h posttransfection, cells were collected and luciferase activities were measured. Luciferase values were adjusted for transfection efficiency using RSV β-Gal. The graph represents the luciferase activity from three independent experiments. The standard deviation for the three experiments is included. (B) Western blot analysis was performed for Tax (Tab172) and CARM1 (Upstate).

    Article Snippet: To examine whether CARM1 plays a role in Tax transactivation of the HTLV-1 LTR, we first compared the relative level of Tax transactivation in the presence and absence of exogenous CARM1.

    Techniques: Mutagenesis, Transfection, Construct, Expressing, Luciferase, Activity Assay, Standard Deviation, Western Blot

    Overexpression of CARM1 increases Tax transcriptional activity of the HTLV-1 LTR. (A to C) HeLa cells were transiently transfected using Effectene transfection reagent (QIAGEN) with reporter construct (0.1 μg) HTLV-1 LTR-Luc (A) or GEM- or GEM(del CREM)-Luc (C), RSV β-Gal (0.05 μg), CARM1 (0.1, 0.2, or 0.4 μg), or Tax (0.1 μg) expression plasmids. At 24 h posttransfection, cells were collected, and luciferase activities were measured. (B) Western blot analysis was performed for Tax (Tab172) and CARM1 (Upstate). (D and E) Jurkat and Molt-4 cells were transfected using Superfect transfection reagent (QIAGEN) with HTLV-1 LTR-Luc (1 μg), RSV β-Gal (0.5 μg), CARM1 (1 or 2 μg), or Tax (1 μg) expression plasmids. All luciferase values were adjusted for transfection efficiency using RSV β-Gal. The graph represents the luciferase activity from three independent experiments. The standard deviation for the three experiments is included.

    Journal: Journal of Virology

    Article Title: Coactivator-Associated Arginine Methyltransferase 1 Enhances Transcriptional Activity of the Human T-Cell Lymphotropic Virus Type 1 Long Terminal Repeat through Direct Interaction with Tax

    doi: 10.1128/JVI.00186-06

    Figure Lengend Snippet: Overexpression of CARM1 increases Tax transcriptional activity of the HTLV-1 LTR. (A to C) HeLa cells were transiently transfected using Effectene transfection reagent (QIAGEN) with reporter construct (0.1 μg) HTLV-1 LTR-Luc (A) or GEM- or GEM(del CREM)-Luc (C), RSV β-Gal (0.05 μg), CARM1 (0.1, 0.2, or 0.4 μg), or Tax (0.1 μg) expression plasmids. At 24 h posttransfection, cells were collected, and luciferase activities were measured. (B) Western blot analysis was performed for Tax (Tab172) and CARM1 (Upstate). (D and E) Jurkat and Molt-4 cells were transfected using Superfect transfection reagent (QIAGEN) with HTLV-1 LTR-Luc (1 μg), RSV β-Gal (0.5 μg), CARM1 (1 or 2 μg), or Tax (1 μg) expression plasmids. All luciferase values were adjusted for transfection efficiency using RSV β-Gal. The graph represents the luciferase activity from three independent experiments. The standard deviation for the three experiments is included.

    Article Snippet: To examine whether CARM1 plays a role in Tax transactivation of the HTLV-1 LTR, we first compared the relative level of Tax transactivation in the presence and absence of exogenous CARM1.

    Techniques: Over Expression, Activity Assay, Transfection, Construct, Expressing, Luciferase, Western Blot, Standard Deviation

    Blocking endogenous CARM1 using siRNA prevents Tax activation of HTLV-1 LTR. (A) siRNA (100 nM) for CARM1 (Santa Cruz Biotechnologies) or CAT (QIAGEN) was transfected with RNAifect reagent (QIAGEN) into HeLa cells. After 12 h, cells were transiently transfected using Effectene transfection reagent with reporter construct HTLV-1 LTR-Luc (0.1 μg), RSV β-Gal (0.05 μg), or Tax (0.1 μg) expression plasmids. At 24 h posttransfection, cells were collected, and luciferase activities were measured. Luciferase values were adjusted for transfection efficiency using RSV β-Gal. The graph represents the luciferase activity from three independent experiments. The standard deviation for the three experiments is included. (B) Western blot analysis was performed for Tax (Tab172) and CARM1 (Upstate).

    Journal: Journal of Virology

    Article Title: Coactivator-Associated Arginine Methyltransferase 1 Enhances Transcriptional Activity of the Human T-Cell Lymphotropic Virus Type 1 Long Terminal Repeat through Direct Interaction with Tax

    doi: 10.1128/JVI.00186-06

    Figure Lengend Snippet: Blocking endogenous CARM1 using siRNA prevents Tax activation of HTLV-1 LTR. (A) siRNA (100 nM) for CARM1 (Santa Cruz Biotechnologies) or CAT (QIAGEN) was transfected with RNAifect reagent (QIAGEN) into HeLa cells. After 12 h, cells were transiently transfected using Effectene transfection reagent with reporter construct HTLV-1 LTR-Luc (0.1 μg), RSV β-Gal (0.05 μg), or Tax (0.1 μg) expression plasmids. At 24 h posttransfection, cells were collected, and luciferase activities were measured. Luciferase values were adjusted for transfection efficiency using RSV β-Gal. The graph represents the luciferase activity from three independent experiments. The standard deviation for the three experiments is included. (B) Western blot analysis was performed for Tax (Tab172) and CARM1 (Upstate).

    Article Snippet: To examine whether CARM1 plays a role in Tax transactivation of the HTLV-1 LTR, we first compared the relative level of Tax transactivation in the presence and absence of exogenous CARM1.

    Techniques: Blocking Assay, Activation Assay, Transfection, Construct, Expressing, Luciferase, Activity Assay, Standard Deviation, Western Blot

    CARM1 and Tax directly interact in vitro and in vivo. (A and B) GST or GST-Tax wild type (A, lanes 1 and 2) and GST-Tax deletion mutants (B) were incubated with purified CARM1 protein. Shown is Western blot analysis of CARM1 levels from GST-Tax pull-down after incubation of HeLa cell nuclear extract with GST or GST-Tax (A, top panel, lanes 3 and 4). Samples were separated by electrophoresis in 4 to 20% Tris-glycine gels, and Western blot analysis for CARM1 or GST was performed. (C) Nuclear extracts from C81 cells were immunoprecipitated (IP) with anti-CARM1 antibody (Ab) and washed extensively, and proteins were separated by electrophoresis in 4 to 20% Tris-glycine gels. Western blot analyses for Tax, CARM1, or p300 were performed. (D) Colocalization of Tax and CARM1. C81 cells were fixed and then immunostained with anti-Tax (α-Tax) and/or anti-CARM1 (α-CARM1) antibodies. (E) 293T cells were transfected with Tax WT or mutant del 151-204, M29, M30, or M32, using Effectene transfection reagent (QIAGEN). At 48 h posttransfection, cell lysates were prepared and immunoprecipated with anti-CARM1 antibody. Immunoprecipitates were separated by electrophoresis in 4 to 20% Tris-glycine gels. Western blot analyses for Tax or CARM1 were performed. The lower panel indicates the levels of expression of Tax and CARM1. (F) Luciferase activities of HTLV-1 LTR were measured in 293T cells transfected with Tax WT or mutants.

    Journal: Journal of Virology

    Article Title: Coactivator-Associated Arginine Methyltransferase 1 Enhances Transcriptional Activity of the Human T-Cell Lymphotropic Virus Type 1 Long Terminal Repeat through Direct Interaction with Tax

    doi: 10.1128/JVI.00186-06

    Figure Lengend Snippet: CARM1 and Tax directly interact in vitro and in vivo. (A and B) GST or GST-Tax wild type (A, lanes 1 and 2) and GST-Tax deletion mutants (B) were incubated with purified CARM1 protein. Shown is Western blot analysis of CARM1 levels from GST-Tax pull-down after incubation of HeLa cell nuclear extract with GST or GST-Tax (A, top panel, lanes 3 and 4). Samples were separated by electrophoresis in 4 to 20% Tris-glycine gels, and Western blot analysis for CARM1 or GST was performed. (C) Nuclear extracts from C81 cells were immunoprecipitated (IP) with anti-CARM1 antibody (Ab) and washed extensively, and proteins were separated by electrophoresis in 4 to 20% Tris-glycine gels. Western blot analyses for Tax, CARM1, or p300 were performed. (D) Colocalization of Tax and CARM1. C81 cells were fixed and then immunostained with anti-Tax (α-Tax) and/or anti-CARM1 (α-CARM1) antibodies. (E) 293T cells were transfected with Tax WT or mutant del 151-204, M29, M30, or M32, using Effectene transfection reagent (QIAGEN). At 48 h posttransfection, cell lysates were prepared and immunoprecipated with anti-CARM1 antibody. Immunoprecipitates were separated by electrophoresis in 4 to 20% Tris-glycine gels. Western blot analyses for Tax or CARM1 were performed. The lower panel indicates the levels of expression of Tax and CARM1. (F) Luciferase activities of HTLV-1 LTR were measured in 293T cells transfected with Tax WT or mutants.

    Article Snippet: To examine whether CARM1 plays a role in Tax transactivation of the HTLV-1 LTR, we first compared the relative level of Tax transactivation in the presence and absence of exogenous CARM1.

    Techniques: In Vitro, In Vivo, Incubation, Purification, Western Blot, Electrophoresis, Immunoprecipitation, Transfection, Mutagenesis, Expressing, Luciferase

    CTAR2 is the major site of LMP1-mediated Fascin induction. (A) Scheme of LMP1 and its signaling domains, the C-terminal activating regions 1 (CTAR1; aa 194–231) and 2 (CTAR2; aa 351–386). Mutations of the P(204)xQxT consensus motif to AxAxA in CTAR1 and deletion of aa 371–386 in CTAR2 are indicated. Signaling pathways induced by LMP1 are shown in rectangular boxes, adaptor molecules in rounded boxes. can. indicates canonical; noncan., noncanonical. (B) qPCR of Fascin transcripts 48 h after transfection of Jurkat cells with wt-LMP1 (20 μg pCMV-HA-LMP1), LMP1 mutants (40 μg pCMV-HA-LMP1(AAA), 20 μg of pCMV-HA-LMP1-Δ371-386) and HTLV-1/Tax (40 μg pcTax-1) in comparison to mock-transfected cells (100 μg pcDNA3). Total transfected DNA was adjusted to 100 μg with pcDNA3. Copy numbers were normalized to those of ACTB and on mock-transfected cells. Mean values +/− standard error (SE) are given. (C) Detection of Fascin and LMP1 by immunoblot after transient transfection of Jurkat cells with the constructs described in (B) . Fascin expression was quantitatively evaluated by densitometry and normalized on LMP1 protein expression. The mean of three independent experiments +/−SE is shown. **indicates P

    Journal: Cell Communication and Signaling : CCS

    Article Title: The tumor marker Fascin is induced by the Epstein-Barr virus-encoded oncoprotein LMP1 via NF-κB in lymphocytes and contributes to their invasive migration

    doi: 10.1186/s12964-014-0046-x

    Figure Lengend Snippet: CTAR2 is the major site of LMP1-mediated Fascin induction. (A) Scheme of LMP1 and its signaling domains, the C-terminal activating regions 1 (CTAR1; aa 194–231) and 2 (CTAR2; aa 351–386). Mutations of the P(204)xQxT consensus motif to AxAxA in CTAR1 and deletion of aa 371–386 in CTAR2 are indicated. Signaling pathways induced by LMP1 are shown in rectangular boxes, adaptor molecules in rounded boxes. can. indicates canonical; noncan., noncanonical. (B) qPCR of Fascin transcripts 48 h after transfection of Jurkat cells with wt-LMP1 (20 μg pCMV-HA-LMP1), LMP1 mutants (40 μg pCMV-HA-LMP1(AAA), 20 μg of pCMV-HA-LMP1-Δ371-386) and HTLV-1/Tax (40 μg pcTax-1) in comparison to mock-transfected cells (100 μg pcDNA3). Total transfected DNA was adjusted to 100 μg with pcDNA3. Copy numbers were normalized to those of ACTB and on mock-transfected cells. Mean values +/− standard error (SE) are given. (C) Detection of Fascin and LMP1 by immunoblot after transient transfection of Jurkat cells with the constructs described in (B) . Fascin expression was quantitatively evaluated by densitometry and normalized on LMP1 protein expression. The mean of three independent experiments +/−SE is shown. **indicates P

    Article Snippet: Total transfected DNA was adjusted to 100 μg with pcDNA3 (Life Technologies GmbH).

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

    SAMHD1 suppresses gene expression driven by the LTR from HIV-1 or HTLV-1 but not from MLV. (A to F) HEK293T cells were transfected with an empty vector (V) or increasing amounts of constructs expressing HA-tagged SAMHD1 and either an HIV-1 LTR-driven FF-Luc construct with (+) or without (−) an HIV-1 Tat-expressing plasmid (A and B), an HTLV-1 LTR-driven FF-Luc construct with or without an HTLV-1 Tax-encoding plasmid (C and D), or an MLV LTR-driven FF-Luc construct (E and F). Overexpression of SAMHD1 was analyzed by immunoblotting (A, C, and E) with GAPDH as a loading control. Cotransfection of Ren-Luc was used as a control of transfection efficiency, with LTR-driven FF-Luc activity normalized to Ren-Luc activity. (B, D, and F) Luciferase activity was determined 24 h posttransfection. Error bars show standard errors of the means for three (HIV-Luc with or without Tat, HTLV-Luc with Tax) or two (HTLV-Luc without Tax, MLV-Luc) independent experiments. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple-comparison posttest. Asterisks indicate significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001) from results for vector (V) control cells.

    Journal: Journal of Virology

    Article Title: SAMHD1 Impairs HIV-1 Gene Expression and Negatively Modulates Reactivation of Viral Latency in CD4+ T Cells

    doi: 10.1128/JVI.00292-18

    Figure Lengend Snippet: SAMHD1 suppresses gene expression driven by the LTR from HIV-1 or HTLV-1 but not from MLV. (A to F) HEK293T cells were transfected with an empty vector (V) or increasing amounts of constructs expressing HA-tagged SAMHD1 and either an HIV-1 LTR-driven FF-Luc construct with (+) or without (−) an HIV-1 Tat-expressing plasmid (A and B), an HTLV-1 LTR-driven FF-Luc construct with or without an HTLV-1 Tax-encoding plasmid (C and D), or an MLV LTR-driven FF-Luc construct (E and F). Overexpression of SAMHD1 was analyzed by immunoblotting (A, C, and E) with GAPDH as a loading control. Cotransfection of Ren-Luc was used as a control of transfection efficiency, with LTR-driven FF-Luc activity normalized to Ren-Luc activity. (B, D, and F) Luciferase activity was determined 24 h posttransfection. Error bars show standard errors of the means for three (HIV-Luc with or without Tat, HTLV-Luc with Tax) or two (HTLV-Luc without Tax, MLV-Luc) independent experiments. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple-comparison posttest. Asterisks indicate significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001) from results for vector (V) control cells.

    Article Snippet: The HIV-1 FF-Luc vector (pGL3-LTR-luc) was provided by Jian-Hua Wang (Pasteur Institute of Shanghai) ( ).

    Techniques: Expressing, Transfection, Plasmid Preparation, Construct, Over Expression, Cotransfection, Activity Assay, Luciferase

    Nonphosphorylated and dNTPase-inactive SAMHD1 mutants show impaired suppression of HIV-1 LTR activity. (A to C) An HIV-1 LTR-driven FF-Luc construct was cotransfected with increasing amounts of plasmids encoding HA-tagged WT SAMHD1, the nonphosphorylated T592A mutant, or the dNTPase-inactive HD/RN mutant into HEK293T cells. Cotransfection of Ren-Luc was used as a control of transfection efficiency. (A) SAMHD1 expression was confirmed by immunoblotting. GAPDH was used as a loading control. Relative SAMHD1 expression levels quantified by densitometry were normalized to GAPDH levels. (B and C) Relative Ren-Luc units were normalized to the total protein concentration (B), and relative FF-Luc units were normalized to Ren-Luc levels (C). Vector cell luciferase activity was set as 1. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple-comparison posttest. Error bars show standard deviations from at least three independent experiments. Asterisks indicate significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001) from results for vector control cells.

    Journal: Journal of Virology

    Article Title: SAMHD1 Impairs HIV-1 Gene Expression and Negatively Modulates Reactivation of Viral Latency in CD4+ T Cells

    doi: 10.1128/JVI.00292-18

    Figure Lengend Snippet: Nonphosphorylated and dNTPase-inactive SAMHD1 mutants show impaired suppression of HIV-1 LTR activity. (A to C) An HIV-1 LTR-driven FF-Luc construct was cotransfected with increasing amounts of plasmids encoding HA-tagged WT SAMHD1, the nonphosphorylated T592A mutant, or the dNTPase-inactive HD/RN mutant into HEK293T cells. Cotransfection of Ren-Luc was used as a control of transfection efficiency. (A) SAMHD1 expression was confirmed by immunoblotting. GAPDH was used as a loading control. Relative SAMHD1 expression levels quantified by densitometry were normalized to GAPDH levels. (B and C) Relative Ren-Luc units were normalized to the total protein concentration (B), and relative FF-Luc units were normalized to Ren-Luc levels (C). Vector cell luciferase activity was set as 1. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple-comparison posttest. Error bars show standard deviations from at least three independent experiments. Asterisks indicate significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001) from results for vector control cells.

    Article Snippet: The HIV-1 FF-Luc vector (pGL3-LTR-luc) was provided by Jian-Hua Wang (Pasteur Institute of Shanghai) ( ).

    Techniques: Activity Assay, Construct, Mutagenesis, Cotransfection, Transfection, Expressing, Protein Concentration, Plasmid Preparation, Luciferase

    SAMHD1 suppresses HIV-1 LTR-driven luciferase expression. (A to E) An HIV-1 LTR-driven firefly luciferase (FF-Luc) construct was cotransfected with an empty vector (V) or increasing amounts of a plasmid encoding HA-tagged SAMHD1 (pSAMHD1) into HEK293T cells. Cotransfection of a construct encoding HSV TK-driven Renilla luciferase (Ren-Luc) was used as a control of transfection efficiency. (A) Overexpression of SAMHD1 was confirmed by immunoblotting. GAPDH was used as a loading control. Relative SAMHD1 expression levels were quantified by densitometry and normalized to GAPDH levels, with 1,000 ng of the pSAMHD1 sample set as 1. (B through E) Ren-Luc activity (B) and mRNA levels (C), and FF-Luc activity (D) and mRNA levels (E), were measured at 24 h posttransfection. (B) Ren-Luc activity was normalized to the total protein concentration. (D and E) FF-Luc activity and mRNA levels were normalized to Ren-Luc activity and mRNA levels, with vector levels set as 1. Error bars show standard deviations for at least three independent experiments as analyzed by one-way ANOVA with Dunnett's multiple-comparison posttest. Asterisks indicate significant differences (****, P ≤ 0.0001) from results for vector control cells. (F to H) FF-Luc and Ren-Luc constructs were expressed by nucleofection in THP-1 control (Ctrl) cells or SAMHD1 knockout (KO) cells. (F) SAMHD1 KO was confirmed by immunoblotting, with GAPDH used as a loading control. (G and H) Luciferase activity was measured at 48 h postnucleofection. Raw Ren-Luc values were normalized to total protein levels (G), and FF-Luc activity was normalized to Ren-Luc activity (H). Asterisks indicate significant differences (**, P ≤ 0.01) from results for control cells.

    Journal: Journal of Virology

    Article Title: SAMHD1 Impairs HIV-1 Gene Expression and Negatively Modulates Reactivation of Viral Latency in CD4+ T Cells

    doi: 10.1128/JVI.00292-18

    Figure Lengend Snippet: SAMHD1 suppresses HIV-1 LTR-driven luciferase expression. (A to E) An HIV-1 LTR-driven firefly luciferase (FF-Luc) construct was cotransfected with an empty vector (V) or increasing amounts of a plasmid encoding HA-tagged SAMHD1 (pSAMHD1) into HEK293T cells. Cotransfection of a construct encoding HSV TK-driven Renilla luciferase (Ren-Luc) was used as a control of transfection efficiency. (A) Overexpression of SAMHD1 was confirmed by immunoblotting. GAPDH was used as a loading control. Relative SAMHD1 expression levels were quantified by densitometry and normalized to GAPDH levels, with 1,000 ng of the pSAMHD1 sample set as 1. (B through E) Ren-Luc activity (B) and mRNA levels (C), and FF-Luc activity (D) and mRNA levels (E), were measured at 24 h posttransfection. (B) Ren-Luc activity was normalized to the total protein concentration. (D and E) FF-Luc activity and mRNA levels were normalized to Ren-Luc activity and mRNA levels, with vector levels set as 1. Error bars show standard deviations for at least three independent experiments as analyzed by one-way ANOVA with Dunnett's multiple-comparison posttest. Asterisks indicate significant differences (****, P ≤ 0.0001) from results for vector control cells. (F to H) FF-Luc and Ren-Luc constructs were expressed by nucleofection in THP-1 control (Ctrl) cells or SAMHD1 knockout (KO) cells. (F) SAMHD1 KO was confirmed by immunoblotting, with GAPDH used as a loading control. (G and H) Luciferase activity was measured at 48 h postnucleofection. Raw Ren-Luc values were normalized to total protein levels (G), and FF-Luc activity was normalized to Ren-Luc activity (H). Asterisks indicate significant differences (**, P ≤ 0.01) from results for control cells.

    Article Snippet: The HIV-1 FF-Luc vector (pGL3-LTR-luc) was provided by Jian-Hua Wang (Pasteur Institute of Shanghai) ( ).

    Techniques: Luciferase, Expressing, Construct, Plasmid Preparation, Cotransfection, Transfection, Over Expression, Activity Assay, Protein Concentration, Knock-Out