luciferase reporter activity assays Search Results


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  • 99
    Millipore luciferase activity
    Luciferase Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 124 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega luciferase activity
    Expression of a dominant negative form of BS69 increases EBNA2 transactivation. Transactivation assays in BJAB cells using EBNA2-GAL4-DNA binding domain fusion proteins and a GAL4 reporter plasmid. Cells were cotransfected with 300 ng of either type 1 GAL4-DBD:EBNA2 (aa 334-487) or type 2 GAL4-DBD:EBNA2 (301-454) constructs, 500 ng of pFRLuc (Gal4 firefly luciferase reporter), 10 ng of pRL-CMV and 1 μg of BS69 (pCI-BS69) or BS69 ΔMYND (pCI-BS69-ΔMYND) expressing plasmids. For each sample, firefly luciferase values were normalised for transfection efficiency using <t>Renilla</t> luciferase values. Results are presented as luciferase activity relative to the pFR-Luc reporter plasmid plus empty vector (pcDNA3.1-GAL4-DBD). BS69 ΔMYND was also transfected in the absence of EBNA2 expressing constructs. Results show the mean of two independent experiments ± standard deviation.
    Luciferase Activity, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 30477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Berthold Technologies luciferase activity
    Expression of a dominant negative form of BS69 increases EBNA2 transactivation. Transactivation assays in BJAB cells using EBNA2-GAL4-DNA binding domain fusion proteins and a GAL4 reporter plasmid. Cells were cotransfected with 300 ng of either type 1 GAL4-DBD:EBNA2 (aa 334-487) or type 2 GAL4-DBD:EBNA2 (301-454) constructs, 500 ng of pFRLuc (Gal4 firefly luciferase reporter), 10 ng of pRL-CMV and 1 μg of BS69 (pCI-BS69) or BS69 ΔMYND (pCI-BS69-ΔMYND) expressing plasmids. For each sample, firefly luciferase values were normalised for transfection efficiency using <t>Renilla</t> luciferase values. Results are presented as luciferase activity relative to the pFR-Luc reporter plasmid plus empty vector (pcDNA3.1-GAL4-DBD). BS69 ΔMYND was also transfected in the absence of EBNA2 expressing constructs. Results show the mean of two independent experiments ± standard deviation.
    Luciferase Activity, supplied by Berthold Technologies, used in various techniques. Bioz Stars score: 94/100, based on 539 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    BioTek Instruments luciferase activity
    Expression of a dominant negative form of BS69 increases EBNA2 transactivation. Transactivation assays in BJAB cells using EBNA2-GAL4-DNA binding domain fusion proteins and a GAL4 reporter plasmid. Cells were cotransfected with 300 ng of either type 1 GAL4-DBD:EBNA2 (aa 334-487) or type 2 GAL4-DBD:EBNA2 (301-454) constructs, 500 ng of pFRLuc (Gal4 firefly luciferase reporter), 10 ng of pRL-CMV and 1 μg of BS69 (pCI-BS69) or BS69 ΔMYND (pCI-BS69-ΔMYND) expressing plasmids. For each sample, firefly luciferase values were normalised for transfection efficiency using <t>Renilla</t> luciferase values. Results are presented as luciferase activity relative to the pFR-Luc reporter plasmid plus empty vector (pcDNA3.1-GAL4-DBD). BS69 ΔMYND was also transfected in the absence of EBNA2 expressing constructs. Results show the mean of two independent experiments ± standard deviation.
    Luciferase Activity, supplied by BioTek Instruments, used in various techniques. Bioz Stars score: 94/100, based on 363 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Turner Designs luciferase activity
    Expression of a dominant negative form of BS69 increases EBNA2 transactivation. Transactivation assays in BJAB cells using EBNA2-GAL4-DNA binding domain fusion proteins and a GAL4 reporter plasmid. Cells were cotransfected with 300 ng of either type 1 GAL4-DBD:EBNA2 (aa 334-487) or type 2 GAL4-DBD:EBNA2 (301-454) constructs, 500 ng of pFRLuc (Gal4 firefly luciferase reporter), 10 ng of pRL-CMV and 1 μg of BS69 (pCI-BS69) or BS69 ΔMYND (pCI-BS69-ΔMYND) expressing plasmids. For each sample, firefly luciferase values were normalised for transfection efficiency using <t>Renilla</t> luciferase values. Results are presented as luciferase activity relative to the pFR-Luc reporter plasmid plus empty vector (pcDNA3.1-GAL4-DBD). BS69 ΔMYND was also transfected in the absence of EBNA2 expressing constructs. Results show the mean of two independent experiments ± standard deviation.
    Luciferase Activity, supplied by Turner Designs, used in various techniques. Bioz Stars score: 94/100, based on 343 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    BMG Labtech luciferase activity
    Expression of a dominant negative form of BS69 increases EBNA2 transactivation. Transactivation assays in BJAB cells using EBNA2-GAL4-DNA binding domain fusion proteins and a GAL4 reporter plasmid. Cells were cotransfected with 300 ng of either type 1 GAL4-DBD:EBNA2 (aa 334-487) or type 2 GAL4-DBD:EBNA2 (301-454) constructs, 500 ng of pFRLuc (Gal4 firefly luciferase reporter), 10 ng of pRL-CMV and 1 μg of BS69 (pCI-BS69) or BS69 ΔMYND (pCI-BS69-ΔMYND) expressing plasmids. For each sample, firefly luciferase values were normalised for transfection efficiency using <t>Renilla</t> luciferase values. Results are presented as luciferase activity relative to the pFR-Luc reporter plasmid plus empty vector (pcDNA3.1-GAL4-DBD). BS69 ΔMYND was also transfected in the absence of EBNA2 expressing constructs. Results show the mean of two independent experiments ± standard deviation.
    Luciferase Activity, supplied by BMG Labtech, used in various techniques. Bioz Stars score: 92/100, based on 273 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson luciferase activity
    Expression of a dominant negative form of BS69 increases EBNA2 transactivation. Transactivation assays in BJAB cells using EBNA2-GAL4-DNA binding domain fusion proteins and a GAL4 reporter plasmid. Cells were cotransfected with 300 ng of either type 1 GAL4-DBD:EBNA2 (aa 334-487) or type 2 GAL4-DBD:EBNA2 (301-454) constructs, 500 ng of pFRLuc (Gal4 firefly luciferase reporter), 10 ng of pRL-CMV and 1 μg of BS69 (pCI-BS69) or BS69 ΔMYND (pCI-BS69-ΔMYND) expressing plasmids. For each sample, firefly luciferase values were normalised for transfection efficiency using <t>Renilla</t> luciferase values. Results are presented as luciferase activity relative to the pFR-Luc reporter plasmid plus empty vector (pcDNA3.1-GAL4-DBD). BS69 ΔMYND was also transfected in the absence of EBNA2 expressing constructs. Results show the mean of two independent experiments ± standard deviation.
    Luciferase Activity, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genecopoeia luciferase activity
    Expression of a dominant negative form of BS69 increases EBNA2 transactivation. Transactivation assays in BJAB cells using EBNA2-GAL4-DNA binding domain fusion proteins and a GAL4 reporter plasmid. Cells were cotransfected with 300 ng of either type 1 GAL4-DBD:EBNA2 (aa 334-487) or type 2 GAL4-DBD:EBNA2 (301-454) constructs, 500 ng of pFRLuc (Gal4 firefly luciferase reporter), 10 ng of pRL-CMV and 1 μg of BS69 (pCI-BS69) or BS69 ΔMYND (pCI-BS69-ΔMYND) expressing plasmids. For each sample, firefly luciferase values were normalised for transfection efficiency using <t>Renilla</t> luciferase values. Results are presented as luciferase activity relative to the pFR-Luc reporter plasmid plus empty vector (pcDNA3.1-GAL4-DBD). BS69 ΔMYND was also transfected in the absence of EBNA2 expressing constructs. Results show the mean of two independent experiments ± standard deviation.
    Luciferase Activity, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 93/100, based on 129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad luciferase activity
    Expression of a dominant negative form of BS69 increases EBNA2 transactivation. Transactivation assays in BJAB cells using EBNA2-GAL4-DNA binding domain fusion proteins and a GAL4 reporter plasmid. Cells were cotransfected with 300 ng of either type 1 GAL4-DBD:EBNA2 (aa 334-487) or type 2 GAL4-DBD:EBNA2 (301-454) constructs, 500 ng of pFRLuc (Gal4 firefly luciferase reporter), 10 ng of pRL-CMV and 1 μg of BS69 (pCI-BS69) or BS69 ΔMYND (pCI-BS69-ΔMYND) expressing plasmids. For each sample, firefly luciferase values were normalised for transfection efficiency using <t>Renilla</t> luciferase values. Results are presented as luciferase activity relative to the pFR-Luc reporter plasmid plus empty vector (pcDNA3.1-GAL4-DBD). BS69 ΔMYND was also transfected in the absence of EBNA2 expressing constructs. Results show the mean of two independent experiments ± standard deviation.
    Luciferase Activity, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Beyotime luciferase activity
    Expression of a dominant negative form of BS69 increases EBNA2 transactivation. Transactivation assays in BJAB cells using EBNA2-GAL4-DNA binding domain fusion proteins and a GAL4 reporter plasmid. Cells were cotransfected with 300 ng of either type 1 GAL4-DBD:EBNA2 (aa 334-487) or type 2 GAL4-DBD:EBNA2 (301-454) constructs, 500 ng of pFRLuc (Gal4 firefly luciferase reporter), 10 ng of pRL-CMV and 1 μg of BS69 (pCI-BS69) or BS69 ΔMYND (pCI-BS69-ΔMYND) expressing plasmids. For each sample, firefly luciferase values were normalised for transfection efficiency using <t>Renilla</t> luciferase values. Results are presented as luciferase activity relative to the pFR-Luc reporter plasmid plus empty vector (pcDNA3.1-GAL4-DBD). BS69 ΔMYND was also transfected in the absence of EBNA2 expressing constructs. Results show the mean of two independent experiments ± standard deviation.
    Luciferase Activity, supplied by Beyotime, used in various techniques. Bioz Stars score: 92/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Promega luciferase activities
    Expression of a dominant negative form of BS69 increases EBNA2 transactivation. Transactivation assays in BJAB cells using EBNA2-GAL4-DNA binding domain fusion proteins and a GAL4 reporter plasmid. Cells were cotransfected with 300 ng of either type 1 GAL4-DBD:EBNA2 (aa 334-487) or type 2 GAL4-DBD:EBNA2 (301-454) constructs, 500 ng of pFRLuc (Gal4 firefly luciferase reporter), 10 ng of pRL-CMV and 1 μg of BS69 (pCI-BS69) or BS69 ΔMYND (pCI-BS69-ΔMYND) expressing plasmids. For each sample, firefly luciferase values were normalised for transfection efficiency using <t>Renilla</t> luciferase values. Results are presented as luciferase activity relative to the pFR-Luc reporter plasmid plus empty vector (pcDNA3.1-GAL4-DBD). BS69 ΔMYND was also transfected in the absence of EBNA2 expressing constructs. Results show the mean of two independent experiments ± standard deviation.
    Luciferase Activities, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 36911 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Biotium luciferase activity
    Expression of a dominant negative form of BS69 increases EBNA2 transactivation. Transactivation assays in BJAB cells using EBNA2-GAL4-DNA binding domain fusion proteins and a GAL4 reporter plasmid. Cells were cotransfected with 300 ng of either type 1 GAL4-DBD:EBNA2 (aa 334-487) or type 2 GAL4-DBD:EBNA2 (301-454) constructs, 500 ng of pFRLuc (Gal4 firefly luciferase reporter), 10 ng of pRL-CMV and 1 μg of BS69 (pCI-BS69) or BS69 ΔMYND (pCI-BS69-ΔMYND) expressing plasmids. For each sample, firefly luciferase values were normalised for transfection efficiency using <t>Renilla</t> luciferase values. Results are presented as luciferase activity relative to the pFR-Luc reporter plasmid plus empty vector (pcDNA3.1-GAL4-DBD). BS69 ΔMYND was also transfected in the absence of EBNA2 expressing constructs. Results show the mean of two independent experiments ± standard deviation.
    Luciferase Activity, supplied by Biotium, used in various techniques. Bioz Stars score: 93/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Turner Designs renilla luciferase activities
    Bcl-2 is an effector of Pim in mediating resistance to MET inhibitors. (A) Expression of indicated proteins was assessed by immunoblot in EBC-1 or MKN45 cells treated with PHA665752 (PHA, 0.1 µM), AZD1208 (AZD, 1 µM), or the combination of both for 48 h. (B) Cell viability was determined by the XTT assay in cells treated with PHA665752 (PHA, 0.1 µM), AZD1208 (AZD, 1 µM), or the combination of both for 72 h. Results shown are the mean ± SD from three independent experiments, each group in four replicates. (C) Expression of indicated proteins was assessed by immunoblot in MKN45 or EBC-1 treated with a nontargeting control siRNA (siC) or a pool of siRNAs targeting MET (siMET) for 48 h. (D) Expression of indicated proteins was assessed by immunoblot in EBC-1 cells treated with PHA665752 (PHA, 0.1 µM), AZD1208 (1 µM), ABT199 (1 µM), ABT737 (1 µM), or the combinations as indicated for 48 h. (E) Cell viability was determined by the XTT assay in cells treated with PHA665752 (PHA, 0.1 µM), AZD1208 (AZD, 1 µM), ABT199 (1 µM), ABT737 (1 µM), or the combinations as indicated for 72 h. Results shown are the mean ± SD from three independent experiments, each group in four replicates. (F) Expression of the indicated proteins was assessed by immunoblot in EBC-1 cells treated with PHA665752 (PHA, 0.1 µM), AZD1208 (1 µM), siRNAs targeting Bcl-2 (1 µM), siRNAs targeting Bad, or the combinations as indicated for 48 h. (G) Cell viability was determined by the XTT assay in cells treated with PHA665752 (PHA, 0.1 µM), AZD1208 (1 µM), siRNAs targeting Bcl-2 (1 µM), siRNAs targeting Bad, or the combinations as indicated for 72 h. Results shown are the mean ± SD from three independent experiments, with four replicates per group. (H) Luciferase activities were determined in cells transfected with a bicistronic luciferase construct phpRL-BCL2-FL-pA for 24 h before treated with PHA665752 (PHA, 0.1 µM), AZD1208 (AZD, 1 µM), or LGB321 (LGB, 1 µM) as indicated for additional 24 h. Relative ratios of firefly/ <t>Renilla</t> luciferase activities are shown. Results shown are the mean ± SD from three independent experiments, each group in three replicates. (I) Cell viability was determined by the XTT assay in EBC-1PHAR cells treated with AZD1208 (1 µM), ABT199 (1 µM), ABT737 (1 µM) or the indicated combinations for 72 h. Results shown are the mean ± SD from three independent experiments, each group in four replicates. (J) Expression of indicated proteins was assessed by immunoblot in cells treated as in (G) for 48 h.
    Renilla Luciferase Activities, supplied by Turner Designs, used in various techniques. Bioz Stars score: 92/100, based on 240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Expression of a dominant negative form of BS69 increases EBNA2 transactivation. Transactivation assays in BJAB cells using EBNA2-GAL4-DNA binding domain fusion proteins and a GAL4 reporter plasmid. Cells were cotransfected with 300 ng of either type 1 GAL4-DBD:EBNA2 (aa 334-487) or type 2 GAL4-DBD:EBNA2 (301-454) constructs, 500 ng of pFRLuc (Gal4 firefly luciferase reporter), 10 ng of pRL-CMV and 1 μg of BS69 (pCI-BS69) or BS69 ΔMYND (pCI-BS69-ΔMYND) expressing plasmids. For each sample, firefly luciferase values were normalised for transfection efficiency using Renilla luciferase values. Results are presented as luciferase activity relative to the pFR-Luc reporter plasmid plus empty vector (pcDNA3.1-GAL4-DBD). BS69 ΔMYND was also transfected in the absence of EBNA2 expressing constructs. Results show the mean of two independent experiments ± standard deviation.

    Journal: bioRxiv

    Article Title: Increased association between Epstein-Barr virus EBNA2 from type 2 strains and the transcriptional repressor BS69 restricts B cell growth

    doi: 10.1101/464131

    Figure Lengend Snippet: Expression of a dominant negative form of BS69 increases EBNA2 transactivation. Transactivation assays in BJAB cells using EBNA2-GAL4-DNA binding domain fusion proteins and a GAL4 reporter plasmid. Cells were cotransfected with 300 ng of either type 1 GAL4-DBD:EBNA2 (aa 334-487) or type 2 GAL4-DBD:EBNA2 (301-454) constructs, 500 ng of pFRLuc (Gal4 firefly luciferase reporter), 10 ng of pRL-CMV and 1 μg of BS69 (pCI-BS69) or BS69 ΔMYND (pCI-BS69-ΔMYND) expressing plasmids. For each sample, firefly luciferase values were normalised for transfection efficiency using Renilla luciferase values. Results are presented as luciferase activity relative to the pFR-Luc reporter plasmid plus empty vector (pcDNA3.1-GAL4-DBD). BS69 ΔMYND was also transfected in the absence of EBNA2 expressing constructs. Results show the mean of two independent experiments ± standard deviation.

    Article Snippet: 20 μl of lysate was assayed for firefly and Renilla luciferase activity using 20 μl of each dual luciferase assay kit reagent (Promega) and a microplate luminometer (LUMIstar Omega, BMG Labtech).

    Techniques: Expressing, Dominant Negative Mutation, Binding Assay, Plasmid Preparation, Construct, Luciferase, Transfection, Activity Assay, Standard Deviation

    Ta CYP 81D5 confers salinity tolerance by promoting Zat12‐mediated ROS signalling pathway, thereby enhancing ROS scavenging. (a) The H 2 O 2 and (b) MDA contents in WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines. (c) ROS levels of WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines by carboxy‐H 2 DCFDA staining. Bar: 0.2 cm. (d) The abundance of Ta CAT , Ta APX , Ta NOX and Ta AOX transcript in roots of WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines. Ta EF 1‐ α (M90077) (Paolacci et al ., 2009 ) was chosen as the endogenous control. Relative transcript abundance of every gene in WT was calculated by giving the value 1. (e) CAT and (f) APX activities of WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines. (g) The transcript abundance of TaZat12 and Ta ZFP 36 in roots of SR 3 and JN 177. Relative transcript abundance of every gene in JN 177 was calculated by giving the value 1. (h) The transcript abundance of TaZat12 and Ta ZFP 36 in roots of WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines. Relative transcript abundance of every gene in WT was calculated by giving the value 1. (i) Y1H assay showing that TaZat12 could bind the A(G/C)T repeats element in Ta APX promoter. Ta APX ‐P : the ~500‐bp promoter fragment of Ta APX ; Ta APX ‐ mP : a fragment deleting the A(G/C)T repeats element; SD /‐Leu, SD medium without Leu; SD /‐Leu/AbA 400 , SD medium without Leu supplemented with AbA at the concentration of 400 ng/mL. Transformed yeast cells were dotted at 10 −1 dilutions on the selective medium. (j) Transient expression assay showing that TaZat12 could activate the expression of Ta APX , and the A(G/C)T repeats element is essential for this activation. The black column represents the relative LUC / REN ratio of the reporter in the present of empty effector ( pBI 221 empty vector) without TaZat12; and the grey column represents the relative LUC / REN ratio of the reporter in the present of effector containing TaZat12. Data are presented as mean ± SE of at least three biological replicates. Columns marked with one asterisk indicate significant differences ( P

    Journal: Plant Biotechnology Journal

    Article Title: TaCYP81D5, one member in a wheat cytochrome P450 gene cluster, confers salinity tolerance via reactive oxygen species scavenging

    doi: 10.1111/pbi.13247

    Figure Lengend Snippet: Ta CYP 81D5 confers salinity tolerance by promoting Zat12‐mediated ROS signalling pathway, thereby enhancing ROS scavenging. (a) The H 2 O 2 and (b) MDA contents in WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines. (c) ROS levels of WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines by carboxy‐H 2 DCFDA staining. Bar: 0.2 cm. (d) The abundance of Ta CAT , Ta APX , Ta NOX and Ta AOX transcript in roots of WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines. Ta EF 1‐ α (M90077) (Paolacci et al ., 2009 ) was chosen as the endogenous control. Relative transcript abundance of every gene in WT was calculated by giving the value 1. (e) CAT and (f) APX activities of WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines. (g) The transcript abundance of TaZat12 and Ta ZFP 36 in roots of SR 3 and JN 177. Relative transcript abundance of every gene in JN 177 was calculated by giving the value 1. (h) The transcript abundance of TaZat12 and Ta ZFP 36 in roots of WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines. Relative transcript abundance of every gene in WT was calculated by giving the value 1. (i) Y1H assay showing that TaZat12 could bind the A(G/C)T repeats element in Ta APX promoter. Ta APX ‐P : the ~500‐bp promoter fragment of Ta APX ; Ta APX ‐ mP : a fragment deleting the A(G/C)T repeats element; SD /‐Leu, SD medium without Leu; SD /‐Leu/AbA 400 , SD medium without Leu supplemented with AbA at the concentration of 400 ng/mL. Transformed yeast cells were dotted at 10 −1 dilutions on the selective medium. (j) Transient expression assay showing that TaZat12 could activate the expression of Ta APX , and the A(G/C)T repeats element is essential for this activation. The black column represents the relative LUC / REN ratio of the reporter in the present of empty effector ( pBI 221 empty vector) without TaZat12; and the grey column represents the relative LUC / REN ratio of the reporter in the present of effector containing TaZat12. Data are presented as mean ± SE of at least three biological replicates. Columns marked with one asterisk indicate significant differences ( P

    Article Snippet: The relative LUC activity was determined by LUC/REN ratio using Dual‐Luciferase Reporter Assay Kit (Promega, Madison, WI) and a Synergy 2 multimode microplate (BioTek, Winooski, VT) according to the manufacturer's instructions.

    Techniques: Multiple Displacement Amplification, Staining, Y1H Assay, Concentration Assay, Transformation Assay, Expressing, Activation Assay, Plasmid Preparation

    Effect of the variants of the rs10892307 LD block. The graphs show the fold of luciferase activity in relation to the empty vector (negative control) of the different constructs corresponding to the four genomic regions with potential regulatory activity containing the four selected CXCR5 polymorphisms: ( A ) SNP rs55756957; ( B ) SNP rs11602393; ( C ) SNP rs10892307; and ( D ) SNP rs3176905. For each region, four constructs carrying either the minor (MA) or the reference (RA) allele and either in forward or in reverse orientation are represented. Each bar represents the mean (± standard deviation) of four independent experiments. Luciferase activity levels are referred to those of an internal control vector containing a basic promoter driving expression of the renilla luciferase reporter protein. LUC: empty plasmid. RA: reference allele. MA: minor allele.

    Journal: Journal of Clinical Medicine

    Article Title: A New Risk Variant for Multiple Sclerosis at 11q23.3 Locus Is Associated with Expansion of CXCR5+ Circulating Regulatory T Cells

    doi: 10.3390/jcm9030625

    Figure Lengend Snippet: Effect of the variants of the rs10892307 LD block. The graphs show the fold of luciferase activity in relation to the empty vector (negative control) of the different constructs corresponding to the four genomic regions with potential regulatory activity containing the four selected CXCR5 polymorphisms: ( A ) SNP rs55756957; ( B ) SNP rs11602393; ( C ) SNP rs10892307; and ( D ) SNP rs3176905. For each region, four constructs carrying either the minor (MA) or the reference (RA) allele and either in forward or in reverse orientation are represented. Each bar represents the mean (± standard deviation) of four independent experiments. Luciferase activity levels are referred to those of an internal control vector containing a basic promoter driving expression of the renilla luciferase reporter protein. LUC: empty plasmid. RA: reference allele. MA: minor allele.

    Article Snippet: After 24 h of incubation in 96-well plates at 37 °C, cells were washed twice in Phosphate Buffered Saline (PBS) at 4 °C and firefly and renilla luciferase activities were evaluated in 40 µg of protein from cell supernatants using Dual Luciferase® Reporter Assay System kit (Promega) and a luminometer F12 (Berthold Detection Systems).

    Techniques: Blocking Assay, Luciferase, Activity Assay, Plasmid Preparation, Negative Control, Construct, Standard Deviation, Expressing

    SOX12 is identified as a direct target gene of HIF-1α. a , b CRC cells were cultured under hypoxic (0.5% O 2 ) conditions for the indicated time intervals and SOX12 expression was examined using qRT-PCR ( a ) and western blotting ( b , c ). A luciferase reporter construct carrying the (−1526/ + 150) SOX12 promoter was transfected into the indicated CRC cells, and luciferase activity was measured after 24 h. ( d ) SW480 cells and SW620 cells were separately infected with HIF-1α lentivirus (LV-HIF-1α) and shHIF-1α lentivirus (LV-shHIF-1α). SOX12 transcription and expression levels were measured after 24 h using luciferase assays (left panel), qRT-PCR (middle panel) and western blotting (right panel). ( e ) Truncated and mutated SOX12 promoter constructs were cotransfected with pCMV-HIF-1α, and the relative luciferase activity was confirmed. ( f ) A ChIP assay confirmed the direct binding of HIF-1α to the SOX12 promoter in CRC cells and human CRC tissues. * P

    Journal: Cell Death & Disease

    Article Title: SOX12 promotes colorectal cancer cell proliferation and metastasis by regulating asparagine synthesis

    doi: 10.1038/s41419-019-1481-9

    Figure Lengend Snippet: SOX12 is identified as a direct target gene of HIF-1α. a , b CRC cells were cultured under hypoxic (0.5% O 2 ) conditions for the indicated time intervals and SOX12 expression was examined using qRT-PCR ( a ) and western blotting ( b , c ). A luciferase reporter construct carrying the (−1526/ + 150) SOX12 promoter was transfected into the indicated CRC cells, and luciferase activity was measured after 24 h. ( d ) SW480 cells and SW620 cells were separately infected with HIF-1α lentivirus (LV-HIF-1α) and shHIF-1α lentivirus (LV-shHIF-1α). SOX12 transcription and expression levels were measured after 24 h using luciferase assays (left panel), qRT-PCR (middle panel) and western blotting (right panel). ( e ) Truncated and mutated SOX12 promoter constructs were cotransfected with pCMV-HIF-1α, and the relative luciferase activity was confirmed. ( f ) A ChIP assay confirmed the direct binding of HIF-1α to the SOX12 promoter in CRC cells and human CRC tissues. * P

    Article Snippet: Luciferase reporter assay Luciferase activity in various treated cells was measured using a Dual Luciferase Assay Kit (Promega, USA) according to the manufacturer’s instructions.

    Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Construct, Transfection, Activity Assay, Infection, Chromatin Immunoprecipitation, Binding Assay

    SOX12 regulates asparagine synthesis by transactivating ASNS , GLS , and GOT2 expression in human CRC. a ASNS, GLS, and GOT2 are three key enzymes in asparagine synthesis. b , c After CRC cells were infected with LV-SOX12 or LV-shSOX12, GLS, GOT2, and ASNS levels were detected using qRT-PCR ( b ) and western blotting ( c , d ). After cotransfection of the luciferase constructs containing the (− 2046/ + 36) GLS, (− 3786/ + 102) GOT2, or (− 1191/ + 111) ASNS promoters with pCMV-SOX12, the relative luciferase activity was determined. e –g) Serially truncated and mutated GLS ( e ), GOT2 ( f ), and ASNS ( g ) promoter plasmids were cotransfected with pCMV-SOX12, and promoter luciferase assays were performed. h – j A ChIP assay revealed direct interactions between SOX12 and the GLS ( h ), GOT2 ( i ), and ASNS ( j ) promoters in CRC cells. k The levels of the indicated intracellular metabolites in SW480, Caco-2, SW620, and LoVo cells were analyzed using LC-MS/MS. * P

    Journal: Cell Death & Disease

    Article Title: SOX12 promotes colorectal cancer cell proliferation and metastasis by regulating asparagine synthesis

    doi: 10.1038/s41419-019-1481-9

    Figure Lengend Snippet: SOX12 regulates asparagine synthesis by transactivating ASNS , GLS , and GOT2 expression in human CRC. a ASNS, GLS, and GOT2 are three key enzymes in asparagine synthesis. b , c After CRC cells were infected with LV-SOX12 or LV-shSOX12, GLS, GOT2, and ASNS levels were detected using qRT-PCR ( b ) and western blotting ( c , d ). After cotransfection of the luciferase constructs containing the (− 2046/ + 36) GLS, (− 3786/ + 102) GOT2, or (− 1191/ + 111) ASNS promoters with pCMV-SOX12, the relative luciferase activity was determined. e –g) Serially truncated and mutated GLS ( e ), GOT2 ( f ), and ASNS ( g ) promoter plasmids were cotransfected with pCMV-SOX12, and promoter luciferase assays were performed. h – j A ChIP assay revealed direct interactions between SOX12 and the GLS ( h ), GOT2 ( i ), and ASNS ( j ) promoters in CRC cells. k The levels of the indicated intracellular metabolites in SW480, Caco-2, SW620, and LoVo cells were analyzed using LC-MS/MS. * P

    Article Snippet: Luciferase reporter assay Luciferase activity in various treated cells was measured using a Dual Luciferase Assay Kit (Promega, USA) according to the manufacturer’s instructions.

    Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, Cotransfection, Luciferase, Construct, Activity Assay, Chromatin Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    LiCl regulates the expression of Six2 at mRNA and protein level. ( A ) pGL3- Six2 promoter-Luciferase construction was simulate by diagram. The Six2 promoter ranging from −2322 to −323 (Six genome sequence) was obtained from NCBI; ( B ) HEK293T cells were co-tra nsfected with pRL-SV40 (renilla control) and pGL3- Six2 promoter-LuC for 36 h. Luciferase activity was normalized to Renilla control. p -values were calculated by Student t -test. Values represents mean values ± SEM of triplicate experiments, *** p

    Journal: International Journal of Molecular Sciences

    Article Title: Six2 Is a Coordinator of LiCl-Induced Cell Proliferation and Apoptosis

    doi: 10.3390/ijms17091504

    Figure Lengend Snippet: LiCl regulates the expression of Six2 at mRNA and protein level. ( A ) pGL3- Six2 promoter-Luciferase construction was simulate by diagram. The Six2 promoter ranging from −2322 to −323 (Six genome sequence) was obtained from NCBI; ( B ) HEK293T cells were co-tra nsfected with pRL-SV40 (renilla control) and pGL3- Six2 promoter-LuC for 36 h. Luciferase activity was normalized to Renilla control. p -values were calculated by Student t -test. Values represents mean values ± SEM of triplicate experiments, *** p

    Article Snippet: For luciferase assays, HEK293T cells were transfected with pGL3-Six2 promoter-luciferase (500 ng/well) and pRL-SV40 (10 ng/well) for 36 h and then treated with LiCl of increasing dosages for 12 h. Luciferase (Luc) activity was assayed using Dual-Luciferase Reporter assay kit (Promega).

    Techniques: Expressing, Luciferase, Sequencing, Activity Assay

    Bcl-2 is an effector of Pim in mediating resistance to MET inhibitors. (A) Expression of indicated proteins was assessed by immunoblot in EBC-1 or MKN45 cells treated with PHA665752 (PHA, 0.1 µM), AZD1208 (AZD, 1 µM), or the combination of both for 48 h. (B) Cell viability was determined by the XTT assay in cells treated with PHA665752 (PHA, 0.1 µM), AZD1208 (AZD, 1 µM), or the combination of both for 72 h. Results shown are the mean ± SD from three independent experiments, each group in four replicates. (C) Expression of indicated proteins was assessed by immunoblot in MKN45 or EBC-1 treated with a nontargeting control siRNA (siC) or a pool of siRNAs targeting MET (siMET) for 48 h. (D) Expression of indicated proteins was assessed by immunoblot in EBC-1 cells treated with PHA665752 (PHA, 0.1 µM), AZD1208 (1 µM), ABT199 (1 µM), ABT737 (1 µM), or the combinations as indicated for 48 h. (E) Cell viability was determined by the XTT assay in cells treated with PHA665752 (PHA, 0.1 µM), AZD1208 (AZD, 1 µM), ABT199 (1 µM), ABT737 (1 µM), or the combinations as indicated for 72 h. Results shown are the mean ± SD from three independent experiments, each group in four replicates. (F) Expression of the indicated proteins was assessed by immunoblot in EBC-1 cells treated with PHA665752 (PHA, 0.1 µM), AZD1208 (1 µM), siRNAs targeting Bcl-2 (1 µM), siRNAs targeting Bad, or the combinations as indicated for 48 h. (G) Cell viability was determined by the XTT assay in cells treated with PHA665752 (PHA, 0.1 µM), AZD1208 (1 µM), siRNAs targeting Bcl-2 (1 µM), siRNAs targeting Bad, or the combinations as indicated for 72 h. Results shown are the mean ± SD from three independent experiments, with four replicates per group. (H) Luciferase activities were determined in cells transfected with a bicistronic luciferase construct phpRL-BCL2-FL-pA for 24 h before treated with PHA665752 (PHA, 0.1 µM), AZD1208 (AZD, 1 µM), or LGB321 (LGB, 1 µM) as indicated for additional 24 h. Relative ratios of firefly/ Renilla luciferase activities are shown. Results shown are the mean ± SD from three independent experiments, each group in three replicates. (I) Cell viability was determined by the XTT assay in EBC-1PHAR cells treated with AZD1208 (1 µM), ABT199 (1 µM), ABT737 (1 µM) or the indicated combinations for 72 h. Results shown are the mean ± SD from three independent experiments, each group in four replicates. (J) Expression of indicated proteins was assessed by immunoblot in cells treated as in (G) for 48 h.

    Journal: Cancer research

    Article Title: Activation of Pim Kinases Is Sufficient to Promote Resistance to MET Small Molecule Inhibitors

    doi: 10.1158/0008-5472.CAN-15-0544

    Figure Lengend Snippet: Bcl-2 is an effector of Pim in mediating resistance to MET inhibitors. (A) Expression of indicated proteins was assessed by immunoblot in EBC-1 or MKN45 cells treated with PHA665752 (PHA, 0.1 µM), AZD1208 (AZD, 1 µM), or the combination of both for 48 h. (B) Cell viability was determined by the XTT assay in cells treated with PHA665752 (PHA, 0.1 µM), AZD1208 (AZD, 1 µM), or the combination of both for 72 h. Results shown are the mean ± SD from three independent experiments, each group in four replicates. (C) Expression of indicated proteins was assessed by immunoblot in MKN45 or EBC-1 treated with a nontargeting control siRNA (siC) or a pool of siRNAs targeting MET (siMET) for 48 h. (D) Expression of indicated proteins was assessed by immunoblot in EBC-1 cells treated with PHA665752 (PHA, 0.1 µM), AZD1208 (1 µM), ABT199 (1 µM), ABT737 (1 µM), or the combinations as indicated for 48 h. (E) Cell viability was determined by the XTT assay in cells treated with PHA665752 (PHA, 0.1 µM), AZD1208 (AZD, 1 µM), ABT199 (1 µM), ABT737 (1 µM), or the combinations as indicated for 72 h. Results shown are the mean ± SD from three independent experiments, each group in four replicates. (F) Expression of the indicated proteins was assessed by immunoblot in EBC-1 cells treated with PHA665752 (PHA, 0.1 µM), AZD1208 (1 µM), siRNAs targeting Bcl-2 (1 µM), siRNAs targeting Bad, or the combinations as indicated for 48 h. (G) Cell viability was determined by the XTT assay in cells treated with PHA665752 (PHA, 0.1 µM), AZD1208 (1 µM), siRNAs targeting Bcl-2 (1 µM), siRNAs targeting Bad, or the combinations as indicated for 72 h. Results shown are the mean ± SD from three independent experiments, with four replicates per group. (H) Luciferase activities were determined in cells transfected with a bicistronic luciferase construct phpRL-BCL2-FL-pA for 24 h before treated with PHA665752 (PHA, 0.1 µM), AZD1208 (AZD, 1 µM), or LGB321 (LGB, 1 µM) as indicated for additional 24 h. Relative ratios of firefly/ Renilla luciferase activities are shown. Results shown are the mean ± SD from three independent experiments, each group in three replicates. (I) Cell viability was determined by the XTT assay in EBC-1PHAR cells treated with AZD1208 (1 µM), ABT199 (1 µM), ABT737 (1 µM) or the indicated combinations for 72 h. Results shown are the mean ± SD from three independent experiments, each group in four replicates. (J) Expression of indicated proteins was assessed by immunoblot in cells treated as in (G) for 48 h.

    Article Snippet: Firefly luciferase and Renilla luciferase activities were measured using a luminometer (Model TD 20/20; Turner Designs) and the reagents provided with the Dual Luciferase Reporter Kit (Promega).

    Techniques: Expressing, XTT Assay, Luciferase, Transfection, Construct

    Overexpression of Pim kinases contributes to increased protein translation in resistant cells through controlling cap-independent translation. (A) De novo protein synthesis was measured by 35 were quantified by normalizing the intensity of green signal to that of the blue signal using the ImageJ software. Results shown are the mean ± SD from three experiments, each group in two replicates. (E, F) Luciferase activities were measured in cells transfected with an HCV IRES or Bcl-2 IRES plasmid and treated with LGB321 (LGB, 1 µM), BEZ235 (0.5 µM), or PP242 (1 µM). The relative ratio of firefly/ Renilla luciferase activity are shown. Results shown are the mean ± SD from three experiments, each group in three replicates. (G) m 7 GTP binding assay was performed in EBC-1 and its resistant subline EBC-1PHAR treated with AZD1208 (1 µM) or BEZ235 (0.5 µM) for 5 h. Elutes and cell lysates were immunoblotted and probed with the indicated antibodies.

    Journal: Cancer research

    Article Title: Activation of Pim Kinases Is Sufficient to Promote Resistance to MET Small Molecule Inhibitors

    doi: 10.1158/0008-5472.CAN-15-0544

    Figure Lengend Snippet: Overexpression of Pim kinases contributes to increased protein translation in resistant cells through controlling cap-independent translation. (A) De novo protein synthesis was measured by 35 were quantified by normalizing the intensity of green signal to that of the blue signal using the ImageJ software. Results shown are the mean ± SD from three experiments, each group in two replicates. (E, F) Luciferase activities were measured in cells transfected with an HCV IRES or Bcl-2 IRES plasmid and treated with LGB321 (LGB, 1 µM), BEZ235 (0.5 µM), or PP242 (1 µM). The relative ratio of firefly/ Renilla luciferase activity are shown. Results shown are the mean ± SD from three experiments, each group in three replicates. (G) m 7 GTP binding assay was performed in EBC-1 and its resistant subline EBC-1PHAR treated with AZD1208 (1 µM) or BEZ235 (0.5 µM) for 5 h. Elutes and cell lysates were immunoblotted and probed with the indicated antibodies.

    Article Snippet: Firefly luciferase and Renilla luciferase activities were measured using a luminometer (Model TD 20/20; Turner Designs) and the reagents provided with the Dual Luciferase Reporter Kit (Promega).

    Techniques: Over Expression, Software, Luciferase, Transfection, Plasmid Preparation, Activity Assay, GTP Binding Assay