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  • 85
    Promega dual reporter luciferase activity
    Dual Reporter Luciferase Activity, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega dual luciferase activity reporter system
    MiR-548c-3p downregulated SMAD2 by binding it. a A total of 1081 genes were searched as the downstream targets of miR-548c-3p in miRNA, TargetScan and miRDB databases. b , c Differentially expressed mRNAs in OS tissues from the GSE12865 and GSE16088 datasets. d Fourteen upregulated mRNAs were selected. e , f SMAD2 was upregulated by knockdown of miR-548c-3p, and it was downregulated by overexpression of miR-548c-3p. g , h <t>Dual-luciferase</t> <t>reporter</t> assay showed that miR-548c-3p bound the G1 region of SMAD2. i , j Knockdown of LINC00266-1 decreased the mRNA and protein levels of SMAD2. k RIP assay showed that LINC00266-1, miR-548c-3p and SMAD2 were enriched in AGO2. * p
    Dual Luciferase Activity Reporter System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 23041 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Promega dual luc reporter assay system
    MiR-548c-3p downregulated SMAD2 by binding it. a A total of 1081 genes were searched as the downstream targets of miR-548c-3p in miRNA, TargetScan and miRDB databases. b , c Differentially expressed mRNAs in OS tissues from the GSE12865 and GSE16088 datasets. d Fourteen upregulated mRNAs were selected. e , f SMAD2 was upregulated by knockdown of miR-548c-3p, and it was downregulated by overexpression of miR-548c-3p. g , h <t>Dual-luciferase</t> <t>reporter</t> assay showed that miR-548c-3p bound the G1 region of SMAD2. i , j Knockdown of LINC00266-1 decreased the mRNA and protein levels of SMAD2. k RIP assay showed that LINC00266-1, miR-548c-3p and SMAD2 were enriched in AGO2. * p
    Dual Luc Reporter Assay System, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 132 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega dual luciferase reporter gene activity assay a dual luciferase reporter assay system kit
    MiR-548c-3p downregulated SMAD2 by binding it. a A total of 1081 genes were searched as the downstream targets of miR-548c-3p in miRNA, TargetScan and miRDB databases. b , c Differentially expressed mRNAs in OS tissues from the GSE12865 and GSE16088 datasets. d Fourteen upregulated mRNAs were selected. e , f SMAD2 was upregulated by knockdown of miR-548c-3p, and it was downregulated by overexpression of miR-548c-3p. g , h <t>Dual-luciferase</t> <t>reporter</t> assay showed that miR-548c-3p bound the G1 region of SMAD2. i , j Knockdown of LINC00266-1 decreased the mRNA and protein levels of SMAD2. k RIP assay showed that LINC00266-1, miR-548c-3p and SMAD2 were enriched in AGO2. * p
    Dual Luciferase Reporter Gene Activity Assay A Dual Luciferase Reporter Assay System Kit, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Promega dual luciferase activity
    MiR-548c-3p downregulated SMAD2 by binding it. a A total of 1081 genes were searched as the downstream targets of miR-548c-3p in miRNA, TargetScan and miRDB databases. b , c Differentially expressed mRNAs in OS tissues from the GSE12865 and GSE16088 datasets. d Fourteen upregulated mRNAs were selected. e , f SMAD2 was upregulated by knockdown of miR-548c-3p, and it was downregulated by overexpression of miR-548c-3p. g , h <t>Dual-luciferase</t> <t>reporter</t> assay showed that miR-548c-3p bound the G1 region of SMAD2. i , j Knockdown of LINC00266-1 decreased the mRNA and protein levels of SMAD2. k RIP assay showed that LINC00266-1, miR-548c-3p and SMAD2 were enriched in AGO2. * p
    Dual Luciferase Activity, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 1011 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega dual luc kit
    Effect of PXR activators on NF-κB-dependent gene expression in <t>U397-3XκB-LUC</t> cells. (A) Left panel, RT-PCR for PXR and GAPDH in <t>U937-3XκB-LUC</t> cells, Hep-G2 cells and human skin fibroblasts (Skin FB). Each lane is amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (B) Luciferase activity after 5 h treatment with the indicated compounds (DMSO, 0.5% (v/v); RIF, 20 μM; HYP, 1 μM; METYR, 250 μM; PCN 20 μM; SULF 1 mM; IKK2-In 10 μM; GT, 3 μM) normalised to LPS + DMSO vehicle activity. Data are the mean and standard deviation of 3 determinations from the same experiment, typical of at least 4 separate experiments. *Significantly different ( P > 95%) reporter gene expression versus LPS-induced DMSO control using Student's t -test (two tailed). (C) Trypan blue exclusion. Upper panels, light micrographs of typical views 5 h after treatment with 300 ng/ml LPS and the indicated compound. Lower panel, percentage trypan blue exclusion in U937-3XκB-LUC cells treated with 300 ng/ml LPS and the indicated concentration of compound for 5 h (note, grey bars are the concentrations employed to examine effects on NF-κB activity in B). Data are the mean of 3 separate incubations from the same experiment, typical of 3 separate experiments. Insol, compound became insoluble when added to culture medium. *Significantly different ( P > 95%) trypan blue exclusion versus LPS-induced vehicle control (DMSO) using Student's t -test (two tailed). Similar results were obtained with compounds without the addition of LPS (data not shown).
    Dual Luc Kit, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Promega dual luciferase reporter activity assay kit
    Effect of PXR activators on NF-κB-dependent gene expression in <t>U397-3XκB-LUC</t> cells. (A) Left panel, RT-PCR for PXR and GAPDH in <t>U937-3XκB-LUC</t> cells, Hep-G2 cells and human skin fibroblasts (Skin FB). Each lane is amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (B) Luciferase activity after 5 h treatment with the indicated compounds (DMSO, 0.5% (v/v); RIF, 20 μM; HYP, 1 μM; METYR, 250 μM; PCN 20 μM; SULF 1 mM; IKK2-In 10 μM; GT, 3 μM) normalised to LPS + DMSO vehicle activity. Data are the mean and standard deviation of 3 determinations from the same experiment, typical of at least 4 separate experiments. *Significantly different ( P > 95%) reporter gene expression versus LPS-induced DMSO control using Student's t -test (two tailed). (C) Trypan blue exclusion. Upper panels, light micrographs of typical views 5 h after treatment with 300 ng/ml LPS and the indicated compound. Lower panel, percentage trypan blue exclusion in U937-3XκB-LUC cells treated with 300 ng/ml LPS and the indicated concentration of compound for 5 h (note, grey bars are the concentrations employed to examine effects on NF-κB activity in B). Data are the mean of 3 separate incubations from the same experiment, typical of 3 separate experiments. Insol, compound became insoluble when added to culture medium. *Significantly different ( P > 95%) trypan blue exclusion versus LPS-induced vehicle control (DMSO) using Student's t -test (two tailed). Similar results were obtained with compounds without the addition of LPS (data not shown).
    Dual Luciferase Reporter Activity Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Beyotime dual luciferase reporter kit
    Effect of PXR activators on NF-κB-dependent gene expression in <t>U397-3XκB-LUC</t> cells. (A) Left panel, RT-PCR for PXR and GAPDH in <t>U937-3XκB-LUC</t> cells, Hep-G2 cells and human skin fibroblasts (Skin FB). Each lane is amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (B) Luciferase activity after 5 h treatment with the indicated compounds (DMSO, 0.5% (v/v); RIF, 20 μM; HYP, 1 μM; METYR, 250 μM; PCN 20 μM; SULF 1 mM; IKK2-In 10 μM; GT, 3 μM) normalised to LPS + DMSO vehicle activity. Data are the mean and standard deviation of 3 determinations from the same experiment, typical of at least 4 separate experiments. *Significantly different ( P > 95%) reporter gene expression versus LPS-induced DMSO control using Student's t -test (two tailed). (C) Trypan blue exclusion. Upper panels, light micrographs of typical views 5 h after treatment with 300 ng/ml LPS and the indicated compound. Lower panel, percentage trypan blue exclusion in U937-3XκB-LUC cells treated with 300 ng/ml LPS and the indicated concentration of compound for 5 h (note, grey bars are the concentrations employed to examine effects on NF-κB activity in B). Data are the mean of 3 separate incubations from the same experiment, typical of 3 separate experiments. Insol, compound became insoluble when added to culture medium. *Significantly different ( P > 95%) trypan blue exclusion versus LPS-induced vehicle control (DMSO) using Student's t -test (two tailed). Similar results were obtained with compounds without the addition of LPS (data not shown).
    Dual Luciferase Reporter Kit, supplied by Beyotime, used in various techniques. Bioz Stars score: 90/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Promega pmirglo dual luciferase reporter
    Effect of PXR activators on NF-κB-dependent gene expression in <t>U397-3XκB-LUC</t> cells. (A) Left panel, RT-PCR for PXR and GAPDH in <t>U937-3XκB-LUC</t> cells, Hep-G2 cells and human skin fibroblasts (Skin FB). Each lane is amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (B) Luciferase activity after 5 h treatment with the indicated compounds (DMSO, 0.5% (v/v); RIF, 20 μM; HYP, 1 μM; METYR, 250 μM; PCN 20 μM; SULF 1 mM; IKK2-In 10 μM; GT, 3 μM) normalised to LPS + DMSO vehicle activity. Data are the mean and standard deviation of 3 determinations from the same experiment, typical of at least 4 separate experiments. *Significantly different ( P > 95%) reporter gene expression versus LPS-induced DMSO control using Student's t -test (two tailed). (C) Trypan blue exclusion. Upper panels, light micrographs of typical views 5 h after treatment with 300 ng/ml LPS and the indicated compound. Lower panel, percentage trypan blue exclusion in U937-3XκB-LUC cells treated with 300 ng/ml LPS and the indicated concentration of compound for 5 h (note, grey bars are the concentrations employed to examine effects on NF-κB activity in B). Data are the mean of 3 separate incubations from the same experiment, typical of 3 separate experiments. Insol, compound became insoluble when added to culture medium. *Significantly different ( P > 95%) trypan blue exclusion versus LPS-induced vehicle control (DMSO) using Student's t -test (two tailed). Similar results were obtained with compounds without the addition of LPS (data not shown).
    Pmirglo Dual Luciferase Reporter, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Vazyme Biotech Co dual luciferase reporter kit
    Effect of PXR activators on NF-κB-dependent gene expression in <t>U397-3XκB-LUC</t> cells. (A) Left panel, RT-PCR for PXR and GAPDH in <t>U937-3XκB-LUC</t> cells, Hep-G2 cells and human skin fibroblasts (Skin FB). Each lane is amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (B) Luciferase activity after 5 h treatment with the indicated compounds (DMSO, 0.5% (v/v); RIF, 20 μM; HYP, 1 μM; METYR, 250 μM; PCN 20 μM; SULF 1 mM; IKK2-In 10 μM; GT, 3 μM) normalised to LPS + DMSO vehicle activity. Data are the mean and standard deviation of 3 determinations from the same experiment, typical of at least 4 separate experiments. *Significantly different ( P > 95%) reporter gene expression versus LPS-induced DMSO control using Student's t -test (two tailed). (C) Trypan blue exclusion. Upper panels, light micrographs of typical views 5 h after treatment with 300 ng/ml LPS and the indicated compound. Lower panel, percentage trypan blue exclusion in U937-3XκB-LUC cells treated with 300 ng/ml LPS and the indicated concentration of compound for 5 h (note, grey bars are the concentrations employed to examine effects on NF-κB activity in B). Data are the mean of 3 separate incubations from the same experiment, typical of 3 separate experiments. Insol, compound became insoluble when added to culture medium. *Significantly different ( P > 95%) trypan blue exclusion versus LPS-induced vehicle control (DMSO) using Student's t -test (two tailed). Similar results were obtained with compounds without the addition of LPS (data not shown).
    Dual Luciferase Reporter Kit, supplied by Vazyme Biotech Co, used in various techniques. Bioz Stars score: 94/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega dual luciferase reporter assaykit
    Effect of PXR activators on NF-κB-dependent gene expression in <t>U397-3XκB-LUC</t> cells. (A) Left panel, RT-PCR for PXR and GAPDH in <t>U937-3XκB-LUC</t> cells, Hep-G2 cells and human skin fibroblasts (Skin FB). Each lane is amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (B) Luciferase activity after 5 h treatment with the indicated compounds (DMSO, 0.5% (v/v); RIF, 20 μM; HYP, 1 μM; METYR, 250 μM; PCN 20 μM; SULF 1 mM; IKK2-In 10 μM; GT, 3 μM) normalised to LPS + DMSO vehicle activity. Data are the mean and standard deviation of 3 determinations from the same experiment, typical of at least 4 separate experiments. *Significantly different ( P > 95%) reporter gene expression versus LPS-induced DMSO control using Student's t -test (two tailed). (C) Trypan blue exclusion. Upper panels, light micrographs of typical views 5 h after treatment with 300 ng/ml LPS and the indicated compound. Lower panel, percentage trypan blue exclusion in U937-3XκB-LUC cells treated with 300 ng/ml LPS and the indicated concentration of compound for 5 h (note, grey bars are the concentrations employed to examine effects on NF-κB activity in B). Data are the mean of 3 separate incubations from the same experiment, typical of 3 separate experiments. Insol, compound became insoluble when added to culture medium. *Significantly different ( P > 95%) trypan blue exclusion versus LPS-induced vehicle control (DMSO) using Student's t -test (two tailed). Similar results were obtained with compounds without the addition of LPS (data not shown).
    Dual Luciferase Reporter Assaykit, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Promega polymerase activity analysis a dual luciferase reporter assay system
    Effect of PXR activators on NF-κB-dependent gene expression in <t>U397-3XκB-LUC</t> cells. (A) Left panel, RT-PCR for PXR and GAPDH in <t>U937-3XκB-LUC</t> cells, Hep-G2 cells and human skin fibroblasts (Skin FB). Each lane is amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (B) Luciferase activity after 5 h treatment with the indicated compounds (DMSO, 0.5% (v/v); RIF, 20 μM; HYP, 1 μM; METYR, 250 μM; PCN 20 μM; SULF 1 mM; IKK2-In 10 μM; GT, 3 μM) normalised to LPS + DMSO vehicle activity. Data are the mean and standard deviation of 3 determinations from the same experiment, typical of at least 4 separate experiments. *Significantly different ( P > 95%) reporter gene expression versus LPS-induced DMSO control using Student's t -test (two tailed). (C) Trypan blue exclusion. Upper panels, light micrographs of typical views 5 h after treatment with 300 ng/ml LPS and the indicated compound. Lower panel, percentage trypan blue exclusion in U937-3XκB-LUC cells treated with 300 ng/ml LPS and the indicated concentration of compound for 5 h (note, grey bars are the concentrations employed to examine effects on NF-κB activity in B). Data are the mean of 3 separate incubations from the same experiment, typical of 3 separate experiments. Insol, compound became insoluble when added to culture medium. *Significantly different ( P > 95%) trypan blue exclusion versus LPS-induced vehicle control (DMSO) using Student's t -test (two tailed). Similar results were obtained with compounds without the addition of LPS (data not shown).
    Polymerase Activity Analysis A Dual Luciferase Reporter Assay System, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega psicheck2 dual luciferase reporter
    Effect of PXR activators on NF-κB-dependent gene expression in <t>U397-3XκB-LUC</t> cells. (A) Left panel, RT-PCR for PXR and GAPDH in <t>U937-3XκB-LUC</t> cells, Hep-G2 cells and human skin fibroblasts (Skin FB). Each lane is amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (B) Luciferase activity after 5 h treatment with the indicated compounds (DMSO, 0.5% (v/v); RIF, 20 μM; HYP, 1 μM; METYR, 250 μM; PCN 20 μM; SULF 1 mM; IKK2-In 10 μM; GT, 3 μM) normalised to LPS + DMSO vehicle activity. Data are the mean and standard deviation of 3 determinations from the same experiment, typical of at least 4 separate experiments. *Significantly different ( P > 95%) reporter gene expression versus LPS-induced DMSO control using Student's t -test (two tailed). (C) Trypan blue exclusion. Upper panels, light micrographs of typical views 5 h after treatment with 300 ng/ml LPS and the indicated compound. Lower panel, percentage trypan blue exclusion in U937-3XκB-LUC cells treated with 300 ng/ml LPS and the indicated concentration of compound for 5 h (note, grey bars are the concentrations employed to examine effects on NF-κB activity in B). Data are the mean of 3 separate incubations from the same experiment, typical of 3 separate experiments. Insol, compound became insoluble when added to culture medium. *Significantly different ( P > 95%) trypan blue exclusion versus LPS-induced vehicle control (DMSO) using Student's t -test (two tailed). Similar results were obtained with compounds without the addition of LPS (data not shown).
    Psicheck2 Dual Luciferase Reporter, supplied by Promega, 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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    93
    Promega pgl3 dual luciferase reporter vectors
    Effect of PXR activators on NF-κB-dependent gene expression in <t>U397-3XκB-LUC</t> cells. (A) Left panel, RT-PCR for PXR and GAPDH in <t>U937-3XκB-LUC</t> cells, Hep-G2 cells and human skin fibroblasts (Skin FB). Each lane is amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (B) Luciferase activity after 5 h treatment with the indicated compounds (DMSO, 0.5% (v/v); RIF, 20 μM; HYP, 1 μM; METYR, 250 μM; PCN 20 μM; SULF 1 mM; IKK2-In 10 μM; GT, 3 μM) normalised to LPS + DMSO vehicle activity. Data are the mean and standard deviation of 3 determinations from the same experiment, typical of at least 4 separate experiments. *Significantly different ( P > 95%) reporter gene expression versus LPS-induced DMSO control using Student's t -test (two tailed). (C) Trypan blue exclusion. Upper panels, light micrographs of typical views 5 h after treatment with 300 ng/ml LPS and the indicated compound. Lower panel, percentage trypan blue exclusion in U937-3XκB-LUC cells treated with 300 ng/ml LPS and the indicated concentration of compound for 5 h (note, grey bars are the concentrations employed to examine effects on NF-κB activity in B). Data are the mean of 3 separate incubations from the same experiment, typical of 3 separate experiments. Insol, compound became insoluble when added to culture medium. *Significantly different ( P > 95%) trypan blue exclusion versus LPS-induced vehicle control (DMSO) using Student's t -test (two tailed). Similar results were obtained with compounds without the addition of LPS (data not shown).
    Pgl3 Dual Luciferase Reporter Vectors, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Genecopoeia dual luciferase reporter system
    Effect of PXR activators on NF-κB-dependent gene expression in <t>U397-3XκB-LUC</t> cells. (A) Left panel, RT-PCR for PXR and GAPDH in <t>U937-3XκB-LUC</t> cells, Hep-G2 cells and human skin fibroblasts (Skin FB). Each lane is amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (B) Luciferase activity after 5 h treatment with the indicated compounds (DMSO, 0.5% (v/v); RIF, 20 μM; HYP, 1 μM; METYR, 250 μM; PCN 20 μM; SULF 1 mM; IKK2-In 10 μM; GT, 3 μM) normalised to LPS + DMSO vehicle activity. Data are the mean and standard deviation of 3 determinations from the same experiment, typical of at least 4 separate experiments. *Significantly different ( P > 95%) reporter gene expression versus LPS-induced DMSO control using Student's t -test (two tailed). (C) Trypan blue exclusion. Upper panels, light micrographs of typical views 5 h after treatment with 300 ng/ml LPS and the indicated compound. Lower panel, percentage trypan blue exclusion in U937-3XκB-LUC cells treated with 300 ng/ml LPS and the indicated concentration of compound for 5 h (note, grey bars are the concentrations employed to examine effects on NF-κB activity in B). Data are the mean of 3 separate incubations from the same experiment, typical of 3 separate experiments. Insol, compound became insoluble when added to culture medium. *Significantly different ( P > 95%) trypan blue exclusion versus LPS-induced vehicle control (DMSO) using Student's t -test (two tailed). Similar results were obtained with compounds without the addition of LPS (data not shown).
    Dual Luciferase Reporter System, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 92/100, based on 97 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    MiR-548c-3p downregulated SMAD2 by binding it. a A total of 1081 genes were searched as the downstream targets of miR-548c-3p in miRNA, TargetScan and miRDB databases. b , c Differentially expressed mRNAs in OS tissues from the GSE12865 and GSE16088 datasets. d Fourteen upregulated mRNAs were selected. e , f SMAD2 was upregulated by knockdown of miR-548c-3p, and it was downregulated by overexpression of miR-548c-3p. g , h Dual-luciferase reporter assay showed that miR-548c-3p bound the G1 region of SMAD2. i , j Knockdown of LINC00266-1 decreased the mRNA and protein levels of SMAD2. k RIP assay showed that LINC00266-1, miR-548c-3p and SMAD2 were enriched in AGO2. * p

    Journal: Cell Death & Disease

    Article Title: LINC00266-1/miR-548c-3p/SMAD2 feedback loop stimulates the development of osteosarcoma

    doi: 10.1038/s41419-020-02764-8

    Figure Lengend Snippet: MiR-548c-3p downregulated SMAD2 by binding it. a A total of 1081 genes were searched as the downstream targets of miR-548c-3p in miRNA, TargetScan and miRDB databases. b , c Differentially expressed mRNAs in OS tissues from the GSE12865 and GSE16088 datasets. d Fourteen upregulated mRNAs were selected. e , f SMAD2 was upregulated by knockdown of miR-548c-3p, and it was downregulated by overexpression of miR-548c-3p. g , h Dual-luciferase reporter assay showed that miR-548c-3p bound the G1 region of SMAD2. i , j Knockdown of LINC00266-1 decreased the mRNA and protein levels of SMAD2. k RIP assay showed that LINC00266-1, miR-548c-3p and SMAD2 were enriched in AGO2. * p

    Article Snippet: Forty-eight hours later, cells were collected for measuring luciferase activity using the dual-luciferase reporter system (Promega, WI, USA).

    Techniques: Binding Assay, Over Expression, Luciferase, Reporter Assay

    SMAD2 bound the promoter region of LINC00266-1. a , b LINC00266-1 was upregulated in OS tissues analyzed in the GSE12865 dataset. c LINC00266-1 was upregulated in OS tissues. d Three binding sites for SMAD2 in the upstream region of LINC00266-1 were predicted by Jaspar. e Overexpression of SMAD2 upregulated the protein expression of SMAD2 in MG63 and U2OS cells. f Overexpression of SMAD2 upregulated LINC00266-1 in MG63, U2OS and 293T cells. g Knockdown of SMAD2 downregulated the protein expression of SMAD2 in MG63 and U2OS cells. h Knockdown of SMAD2 downregulated LINC00266-1 in MG63, U2OS and 293T cells. i ChIP assay revealed the binding of SMAD2 to the promoter region of LINC00266-1. j , k Dual-luciferase reporter assay revealed that SMAD2 bound in the region 1328 nucleotides upstream of LINC00266-1. l , m The luciferase reporter plasmid with the mutation sequence of E2 markedly reduced the luciferase activity in the Mutation sequence. * p

    Journal: Cell Death & Disease

    Article Title: LINC00266-1/miR-548c-3p/SMAD2 feedback loop stimulates the development of osteosarcoma

    doi: 10.1038/s41419-020-02764-8

    Figure Lengend Snippet: SMAD2 bound the promoter region of LINC00266-1. a , b LINC00266-1 was upregulated in OS tissues analyzed in the GSE12865 dataset. c LINC00266-1 was upregulated in OS tissues. d Three binding sites for SMAD2 in the upstream region of LINC00266-1 were predicted by Jaspar. e Overexpression of SMAD2 upregulated the protein expression of SMAD2 in MG63 and U2OS cells. f Overexpression of SMAD2 upregulated LINC00266-1 in MG63, U2OS and 293T cells. g Knockdown of SMAD2 downregulated the protein expression of SMAD2 in MG63 and U2OS cells. h Knockdown of SMAD2 downregulated LINC00266-1 in MG63, U2OS and 293T cells. i ChIP assay revealed the binding of SMAD2 to the promoter region of LINC00266-1. j , k Dual-luciferase reporter assay revealed that SMAD2 bound in the region 1328 nucleotides upstream of LINC00266-1. l , m The luciferase reporter plasmid with the mutation sequence of E2 markedly reduced the luciferase activity in the Mutation sequence. * p

    Article Snippet: Forty-eight hours later, cells were collected for measuring luciferase activity using the dual-luciferase reporter system (Promega, WI, USA).

    Techniques: Binding Assay, Over Expression, Expressing, Chromatin Immunoprecipitation, Luciferase, Reporter Assay, Plasmid Preparation, Mutagenesis, Sequencing, Activity Assay

    LINC00266-1 sponged miR-548c-3p and miR-548c-3p abolished the effect of LINC00266-1 on OS cell phenotypes. a LINC00266-1 was distributed in both the cytoplasm and nuclei. b Eleven potential miRNAs binding LINC00266-1 were searched in the Diana and miRDB databases. Literature review showed that five miRNAs have been extensively explored. c MiR-548c-3p was downregulated in OS tissues. d Transfection efficiency of miR-548c-3p mimic and inhibitor. e MiR-548c-3p was upregulated in OS cells with LINC00266-1 knockdown. f LINC00266-1 was downregulated by overexpression of miR-548c-3p, and it was upregulated by knockdown of miR-548c-3p. g – i Dual-luciferase reporter assay showed that the B1 mutation failed to affect the luciferase activity in MT-1, MT-2 and MT-3 in MG63 and U2OS cells. j – l The proliferation, metastasis and apoptosis in MG63 cells were influenced by miR-548c-3p and LINC00266-1. m Relative levels of Bax, Ki67, MMP2 and MMP9 in MG63 cells were influenced by miR-548c-3p and LINC00266-1. n – p The proliferation, metastasis and apoptosis in U2OS cells were influenced by miR-548c-3p and LINC00266-1. q Relative levels of Bax, Ki67, MMP2 and MMP9 in U2OS cells were influenced by miR-548c-3p and LINC00266-1 (* p

    Journal: Cell Death & Disease

    Article Title: LINC00266-1/miR-548c-3p/SMAD2 feedback loop stimulates the development of osteosarcoma

    doi: 10.1038/s41419-020-02764-8

    Figure Lengend Snippet: LINC00266-1 sponged miR-548c-3p and miR-548c-3p abolished the effect of LINC00266-1 on OS cell phenotypes. a LINC00266-1 was distributed in both the cytoplasm and nuclei. b Eleven potential miRNAs binding LINC00266-1 were searched in the Diana and miRDB databases. Literature review showed that five miRNAs have been extensively explored. c MiR-548c-3p was downregulated in OS tissues. d Transfection efficiency of miR-548c-3p mimic and inhibitor. e MiR-548c-3p was upregulated in OS cells with LINC00266-1 knockdown. f LINC00266-1 was downregulated by overexpression of miR-548c-3p, and it was upregulated by knockdown of miR-548c-3p. g – i Dual-luciferase reporter assay showed that the B1 mutation failed to affect the luciferase activity in MT-1, MT-2 and MT-3 in MG63 and U2OS cells. j – l The proliferation, metastasis and apoptosis in MG63 cells were influenced by miR-548c-3p and LINC00266-1. m Relative levels of Bax, Ki67, MMP2 and MMP9 in MG63 cells were influenced by miR-548c-3p and LINC00266-1. n – p The proliferation, metastasis and apoptosis in U2OS cells were influenced by miR-548c-3p and LINC00266-1. q Relative levels of Bax, Ki67, MMP2 and MMP9 in U2OS cells were influenced by miR-548c-3p and LINC00266-1 (* p

    Article Snippet: Forty-eight hours later, cells were collected for measuring luciferase activity using the dual-luciferase reporter system (Promega, WI, USA).

    Techniques: Binding Assay, Transfection, Over Expression, Luciferase, Reporter Assay, Mutagenesis, Activity Assay

    Effect of PXR activators on NF-κB-dependent gene expression in U397-3XκB-LUC cells. (A) Left panel, RT-PCR for PXR and GAPDH in U937-3XκB-LUC cells, Hep-G2 cells and human skin fibroblasts (Skin FB). Each lane is amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (B) Luciferase activity after 5 h treatment with the indicated compounds (DMSO, 0.5% (v/v); RIF, 20 μM; HYP, 1 μM; METYR, 250 μM; PCN 20 μM; SULF 1 mM; IKK2-In 10 μM; GT, 3 μM) normalised to LPS + DMSO vehicle activity. Data are the mean and standard deviation of 3 determinations from the same experiment, typical of at least 4 separate experiments. *Significantly different ( P > 95%) reporter gene expression versus LPS-induced DMSO control using Student's t -test (two tailed). (C) Trypan blue exclusion. Upper panels, light micrographs of typical views 5 h after treatment with 300 ng/ml LPS and the indicated compound. Lower panel, percentage trypan blue exclusion in U937-3XκB-LUC cells treated with 300 ng/ml LPS and the indicated concentration of compound for 5 h (note, grey bars are the concentrations employed to examine effects on NF-κB activity in B). Data are the mean of 3 separate incubations from the same experiment, typical of 3 separate experiments. Insol, compound became insoluble when added to culture medium. *Significantly different ( P > 95%) trypan blue exclusion versus LPS-induced vehicle control (DMSO) using Student's t -test (two tailed). Similar results were obtained with compounds without the addition of LPS (data not shown).

    Journal: The Journal of Steroid Biochemistry and Molecular Biology

    Article Title: The PXR is a drug target for chronic inflammatory liver disease

    doi: 10.1016/j.jsbmb.2010.04.012

    Figure Lengend Snippet: Effect of PXR activators on NF-κB-dependent gene expression in U397-3XκB-LUC cells. (A) Left panel, RT-PCR for PXR and GAPDH in U937-3XκB-LUC cells, Hep-G2 cells and human skin fibroblasts (Skin FB). Each lane is amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (B) Luciferase activity after 5 h treatment with the indicated compounds (DMSO, 0.5% (v/v); RIF, 20 μM; HYP, 1 μM; METYR, 250 μM; PCN 20 μM; SULF 1 mM; IKK2-In 10 μM; GT, 3 μM) normalised to LPS + DMSO vehicle activity. Data are the mean and standard deviation of 3 determinations from the same experiment, typical of at least 4 separate experiments. *Significantly different ( P > 95%) reporter gene expression versus LPS-induced DMSO control using Student's t -test (two tailed). (C) Trypan blue exclusion. Upper panels, light micrographs of typical views 5 h after treatment with 300 ng/ml LPS and the indicated compound. Lower panel, percentage trypan blue exclusion in U937-3XκB-LUC cells treated with 300 ng/ml LPS and the indicated concentration of compound for 5 h (note, grey bars are the concentrations employed to examine effects on NF-κB activity in B). Data are the mean of 3 separate incubations from the same experiment, typical of 3 separate experiments. Insol, compound became insoluble when added to culture medium. *Significantly different ( P > 95%) trypan blue exclusion versus LPS-induced vehicle control (DMSO) using Student's t -test (two tailed). Similar results were obtained with compounds without the addition of LPS (data not shown).

    Article Snippet: Luciferase expression in U937-3XκB-LUC cells stably transfected with luciferase was determined also using the Dual Luc kit.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Luciferase, Activity Assay, Standard Deviation, Two Tailed Test, Concentration Assay

    CsA is an activator of the human PXR. (A) Hep-G2 cells cultured in 24-well plates were transfected with 0.5 μg (ER6) 3 -pGL3promoter and 0.05 μg RL-TK/well and PXR activation determined [6] . Data are the mean and standard deviation of at least 3 separate transfections from the same experiment expressed as fold normalised luciferase expression versus DMSO vehicle control, typical of at least 3 separate experiments. *Significantly different ( P > 95%) luciferase activity versus DMSO using Student's t -test (two tailed). (B) Human hepatocytes were isolated and cultured as outlined in Methods section and treated with the indicated compounds after the first 24 h of culture ( T 0 ) for a further 48 h prior to isolation and analysis of CYP3A4 and β-actin levels by Western blotting. Medium and treatments were replenished every 24 h. (C) RT-PCR for the indicated transcripts in human liver, two separate human hepatocyte preparations (freshly isolated cells) and two separate LS180 cultures (amplified product from the equivalent 100 ng template total RNA per well, 35 PCR cycles). (D) Expression of CYP3A4 mRNA in LS180 cells. LS180 cells were treated with the indicated compound DMSO, 0.1% (v/v); RIF, 20 μM RIF (for 48 h) and CsA and FK506 at the indicated concentrations for 72 h. Culture media and treatments were changed daily. RNA was isolated and transcript CYP3A4 and 18S rRNA levels determined by quantitative RT-PCR. *Significantly different ( P > 95%) transcript level activity versus DMSO using Student's t -test (two tailed).

    Journal: The Journal of Steroid Biochemistry and Molecular Biology

    Article Title: The PXR is a drug target for chronic inflammatory liver disease

    doi: 10.1016/j.jsbmb.2010.04.012

    Figure Lengend Snippet: CsA is an activator of the human PXR. (A) Hep-G2 cells cultured in 24-well plates were transfected with 0.5 μg (ER6) 3 -pGL3promoter and 0.05 μg RL-TK/well and PXR activation determined [6] . Data are the mean and standard deviation of at least 3 separate transfections from the same experiment expressed as fold normalised luciferase expression versus DMSO vehicle control, typical of at least 3 separate experiments. *Significantly different ( P > 95%) luciferase activity versus DMSO using Student's t -test (two tailed). (B) Human hepatocytes were isolated and cultured as outlined in Methods section and treated with the indicated compounds after the first 24 h of culture ( T 0 ) for a further 48 h prior to isolation and analysis of CYP3A4 and β-actin levels by Western blotting. Medium and treatments were replenished every 24 h. (C) RT-PCR for the indicated transcripts in human liver, two separate human hepatocyte preparations (freshly isolated cells) and two separate LS180 cultures (amplified product from the equivalent 100 ng template total RNA per well, 35 PCR cycles). (D) Expression of CYP3A4 mRNA in LS180 cells. LS180 cells were treated with the indicated compound DMSO, 0.1% (v/v); RIF, 20 μM RIF (for 48 h) and CsA and FK506 at the indicated concentrations for 72 h. Culture media and treatments were changed daily. RNA was isolated and transcript CYP3A4 and 18S rRNA levels determined by quantitative RT-PCR. *Significantly different ( P > 95%) transcript level activity versus DMSO using Student's t -test (two tailed).

    Article Snippet: Luciferase expression in U937-3XκB-LUC cells stably transfected with luciferase was determined also using the Dual Luc kit.

    Techniques: Cell Culture, Transfection, Activation Assay, Standard Deviation, Luciferase, Expressing, Activity Assay, Two Tailed Test, Isolation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Quantitative RT-PCR