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  • 93
    Addgene inc prl tk
    TPX blocked MAPK phosphorylation and inhibited NF‐κB activity in LPS‐treated RAW264.7 macrophages. Cells were treated with TPX in the absence or presence of LPS (1 μg · mL −1 ) for the indicated time. (A) The protein levels of phospho‐ERK, ERK, phospho‐p38, p38, phospho‐JNK and JNK were detected by Western blot analyses ( n = 6). α‐Tubulin was used as internal loading control. Data were normalized to the mean value of the LPS group. (B) The protein levels of phospho‐IKKα/β, IKKα, IKKβ, phospho‐IκBα, IκBα, phospho‐p65 and p65 were detected by Western blot analyses ( n = 6). α‐Tubulin was used as internal loading control. Data were normalized to the mean value of the LPS group. (C) Cells were pretreated with or without TPX (20 μM) for 1 h and then exposed to LPS (1 μg · mL −1 ) for another 1 h. The immunostaining of p65 was detected. Nuclei were stained with DAPI. (D) The percentage of cells undergoing p65 nuclear translocation was quantified ( n = 6). (E) The protein level of p65 in nuclear and cytoplasmic fractions was determined by Western blotting. Histone 3 and α‐tubulin were used as internal loading controls. (F) The relative expression of p65 in the nuclear fraction was quantified ( n = 6). Data are normalized to the mean value of LPS group. (G) RAW264.7 cells were co‐transfected with a NF‐kB luciferase reporter gene and <t>pRL</t> Renilla luciferase control reporter vector, then the cells were pretreated with TPX for 1 h followed by activation with LPS for 1 h. The transcriptional activity was expressed as the ratio of firefly to Renilla luciferase intensities in the cell lysates ( n = 6). Data are expressed as means ± SEM. # P
    Prl Tk, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Promega prl tk
    Inhibitory effects of G0S2 domains in different cell contexts. Transient transfection of G0S2 decreased the luciferase activity of the co-transfected <t>PGL3E</t> (firefly, left panel) and <t>pRL-TK</t> (renilla, right panel) reporter plasmids in (A) A549 cells and independently in (B) ED-1 cells. Transient transfection of G0S2 that lacked the HD domain (ΔHD) exhibited significantly less (P
    Prl Tk, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 14463 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa prl tk
    TAK1 inhibition of HBV replication and gene expression requires activation of downstream MAPK pathways. ( A ) Huh7 cells were co-transfected with 1.5 μg of pHBV1.3 and 1.5 μg of pXJ40-HA, or plasmids expressing HA-hTAK1, DN-hTAK1 or CA-hTAK1. Cells were harvested 72 h after transfection. HBV RI, viral RNAs and HBsAg and HBeAg were determined as described above. HA-tag and β-actin (loading control) were examined by Western blot, 28 S/18 S rRNAs served as loading controls for Northern blot analysis. ( B ) Huh7 cells were co-transfected with 100 ng of pCP or pXP, and 100 ng of pXJ40-HA or plasmids expressing HA-hTAK1, DN-TAK1 or CA-TAK1. Cells were harvested 48 h after transfection and firefly and Renilla luciferase activities were measured and normalized against those generated by the pGL3-basic control plasmid. ( C ) Huh7 cells were transfected with 1.5 μg of pXJ40-HA or plasmids expressing HA-hTAK1, DN-TAK1, and CA-TAK1. Phosphorylation and basal expression levels of p38, JNK, ERK, and NF-κBp 65 were analyzed by Western blot 12 h after transfection, using β-actin as loading control. ( D ) Huh7 cells were seeded in 24-well plates and transfected with 100 ng of the reporter plasmids <t>pNF-κB-luc</t> (pNF-kB) or pAP1-luc (pAP-1) and 100 ng of pXJ40-HA, hTAK1, DN-TAK1, or CA-TAK1. For each transfection, 100 ng of <t>pRL-TK</t> was included as an internal control of transfection efficiency. Cells were harvested 48 h after transfection and firefly and Renilla luciferase activities were measured and normalized against those generated by the pGL3-basic control plasmid.
    Prl Tk, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 184 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Ribobio prl tk
    TAK1 inhibition of HBV replication and gene expression requires activation of downstream MAPK pathways. ( A ) Huh7 cells were co-transfected with 1.5 μg of pHBV1.3 and 1.5 μg of pXJ40-HA, or plasmids expressing HA-hTAK1, DN-hTAK1 or CA-hTAK1. Cells were harvested 72 h after transfection. HBV RI, viral RNAs and HBsAg and HBeAg were determined as described above. HA-tag and β-actin (loading control) were examined by Western blot, 28 S/18 S rRNAs served as loading controls for Northern blot analysis. ( B ) Huh7 cells were co-transfected with 100 ng of pCP or pXP, and 100 ng of pXJ40-HA or plasmids expressing HA-hTAK1, DN-TAK1 or CA-TAK1. Cells were harvested 48 h after transfection and firefly and Renilla luciferase activities were measured and normalized against those generated by the pGL3-basic control plasmid. ( C ) Huh7 cells were transfected with 1.5 μg of pXJ40-HA or plasmids expressing HA-hTAK1, DN-TAK1, and CA-TAK1. Phosphorylation and basal expression levels of p38, JNK, ERK, and NF-κBp 65 were analyzed by Western blot 12 h after transfection, using β-actin as loading control. ( D ) Huh7 cells were seeded in 24-well plates and transfected with 100 ng of the reporter plasmids <t>pNF-κB-luc</t> (pNF-kB) or pAP1-luc (pAP-1) and 100 ng of pXJ40-HA, hTAK1, DN-TAK1, or CA-TAK1. For each transfection, 100 ng of <t>pRL-TK</t> was included as an internal control of transfection efficiency. Cells were harvested 48 h after transfection and firefly and Renilla luciferase activities were measured and normalized against those generated by the pGL3-basic control plasmid.
    Prl Tk, supplied by Ribobio, used in various techniques. Bioz Stars score: 92/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche prl tk
    TAK1 inhibition of HBV replication and gene expression requires activation of downstream MAPK pathways. ( A ) Huh7 cells were co-transfected with 1.5 μg of pHBV1.3 and 1.5 μg of pXJ40-HA, or plasmids expressing HA-hTAK1, DN-hTAK1 or CA-hTAK1. Cells were harvested 72 h after transfection. HBV RI, viral RNAs and HBsAg and HBeAg were determined as described above. HA-tag and β-actin (loading control) were examined by Western blot, 28 S/18 S rRNAs served as loading controls for Northern blot analysis. ( B ) Huh7 cells were co-transfected with 100 ng of pCP or pXP, and 100 ng of pXJ40-HA or plasmids expressing HA-hTAK1, DN-TAK1 or CA-TAK1. Cells were harvested 48 h after transfection and firefly and Renilla luciferase activities were measured and normalized against those generated by the pGL3-basic control plasmid. ( C ) Huh7 cells were transfected with 1.5 μg of pXJ40-HA or plasmids expressing HA-hTAK1, DN-TAK1, and CA-TAK1. Phosphorylation and basal expression levels of p38, JNK, ERK, and NF-κBp 65 were analyzed by Western blot 12 h after transfection, using β-actin as loading control. ( D ) Huh7 cells were seeded in 24-well plates and transfected with 100 ng of the reporter plasmids <t>pNF-κB-luc</t> (pNF-kB) or pAP1-luc (pAP-1) and 100 ng of pXJ40-HA, hTAK1, DN-TAK1, or CA-TAK1. For each transfection, 100 ng of <t>pRL-TK</t> was included as an internal control of transfection efficiency. Cells were harvested 48 h after transfection and firefly and Renilla luciferase activities were measured and normalized against those generated by the pGL3-basic control plasmid.
    Prl Tk, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 359 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene prl tk
    Activity-induced reporter gene expression in <t>N2A</t> cells. For KC1 stimulation experiments, tetR-VP16-GS-hNFAT was co-expressed with tetO-Fluc and <t>pRL-TK.</t> When stimulated with 60 mM KC1, the FLuc/RLuc ratio increased nearly 2-fold. For light stimulation experiments, Gal4-NFATAD was co-expressed with VChR1, UAS-Fluc (pFR-Luc), and pRL-TK. After 488-nm light illumination for 12 hours, the Fluc/Rluc ratio increased by about 5-fold. Each data point represents average results from two samples.
    Prl Tk, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher prl tk
    Regions both 5′ and 3′ to the start of transcription were required for SF-1 promoter activity. A region of the Ftz-F1 gene from −734 to +60 bp, relative to the SF-1 transcriptional start site (+1), was cloned upstream of the firefly luciferase reporter gene. Promoter activity was determined by transient transfection analysis in either primary rat Sertoli cells, MSC-1 cells, or MA-10 cells as described in Material and Methods. Promoter constructs were cotransfected with <t>pRL-TK,</t> which expresses <t>Renilla</t> luciferase from the thymidine kinase promoter, and was used to control for transfection efficiency. In both A and B, firefly luciferase activity from the different promoter constructs was normalized to Renilla luciferase values. In A, the firefly/ Renilla luciferase activity of the SF-1 (−734/+60) construct was assayed in each of the cell lines and reported as the activity relative to the firefly/ Renilla luciferase activity of the promoterless control vector pGL3-Basic. In B, various promoter deletion mutants are indicated; the activity of each, determined as described above, was analyzed in each of the cell types. Here, the data represent the firefly/ Renilla luciferase activity of each SF-1 promoter made relative to the firefly/ Renilla luciferase activity of the SF-1 (−734/+60) Luc construct. Transfections were done a minimum of three times. Error bars represent the SEM.
    Prl Tk, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2191 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Upstate Biotechnology Inc prl tk
    Regions both 5′ and 3′ to the start of transcription were required for SF-1 promoter activity. A region of the Ftz-F1 gene from −734 to +60 bp, relative to the SF-1 transcriptional start site (+1), was cloned upstream of the firefly luciferase reporter gene. Promoter activity was determined by transient transfection analysis in either primary rat Sertoli cells, MSC-1 cells, or MA-10 cells as described in Material and Methods. Promoter constructs were cotransfected with <t>pRL-TK,</t> which expresses <t>Renilla</t> luciferase from the thymidine kinase promoter, and was used to control for transfection efficiency. In both A and B, firefly luciferase activity from the different promoter constructs was normalized to Renilla luciferase values. In A, the firefly/ Renilla luciferase activity of the SF-1 (−734/+60) construct was assayed in each of the cell lines and reported as the activity relative to the firefly/ Renilla luciferase activity of the promoterless control vector pGL3-Basic. In B, various promoter deletion mutants are indicated; the activity of each, determined as described above, was analyzed in each of the cell types. Here, the data represent the firefly/ Renilla luciferase activity of each SF-1 promoter made relative to the firefly/ Renilla luciferase activity of the SF-1 (−734/+60) Luc construct. Transfections were done a minimum of three times. Error bars represent the SEM.
    Prl Tk, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Biowit Technologies prl tk
    Regions both 5′ and 3′ to the start of transcription were required for SF-1 promoter activity. A region of the Ftz-F1 gene from −734 to +60 bp, relative to the SF-1 transcriptional start site (+1), was cloned upstream of the firefly luciferase reporter gene. Promoter activity was determined by transient transfection analysis in either primary rat Sertoli cells, MSC-1 cells, or MA-10 cells as described in Material and Methods. Promoter constructs were cotransfected with <t>pRL-TK,</t> which expresses <t>Renilla</t> luciferase from the thymidine kinase promoter, and was used to control for transfection efficiency. In both A and B, firefly luciferase activity from the different promoter constructs was normalized to Renilla luciferase values. In A, the firefly/ Renilla luciferase activity of the SF-1 (−734/+60) construct was assayed in each of the cell lines and reported as the activity relative to the firefly/ Renilla luciferase activity of the promoterless control vector pGL3-Basic. In B, various promoter deletion mutants are indicated; the activity of each, determined as described above, was analyzed in each of the cell types. Here, the data represent the firefly/ Renilla luciferase activity of each SF-1 promoter made relative to the firefly/ Renilla luciferase activity of the SF-1 (−734/+60) Luc construct. Transfections were done a minimum of three times. Error bars represent the SEM.
    Prl Tk, supplied by Biowit Technologies, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega coreporter prl tk
    Regions both 5′ and 3′ to the start of transcription were required for SF-1 promoter activity. A region of the Ftz-F1 gene from −734 to +60 bp, relative to the SF-1 transcriptional start site (+1), was cloned upstream of the firefly luciferase reporter gene. Promoter activity was determined by transient transfection analysis in either primary rat Sertoli cells, MSC-1 cells, or MA-10 cells as described in Material and Methods. Promoter constructs were cotransfected with <t>pRL-TK,</t> which expresses <t>Renilla</t> luciferase from the thymidine kinase promoter, and was used to control for transfection efficiency. In both A and B, firefly luciferase activity from the different promoter constructs was normalized to Renilla luciferase values. In A, the firefly/ Renilla luciferase activity of the SF-1 (−734/+60) construct was assayed in each of the cell lines and reported as the activity relative to the firefly/ Renilla luciferase activity of the promoterless control vector pGL3-Basic. In B, various promoter deletion mutants are indicated; the activity of each, determined as described above, was analyzed in each of the cell types. Here, the data represent the firefly/ Renilla luciferase activity of each SF-1 promoter made relative to the firefly/ Renilla luciferase activity of the SF-1 (−734/+60) Luc construct. Transfections were done a minimum of three times. Error bars represent the SEM.
    Coreporter Prl Tk, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega prl tk reporter
    Inhibition by γ-PGA of SARS-CoV replicon replication. (A) The cytotoxicity of γ-PGA (MW: 2000 kDa) in HEK293hTLR4 cells transiently expressing MD2 and CD14 was evaluated by the MTS assay. Cells were treated with increasing concentrations of γ-PGA for 30 h. Data are expressed as the percentage of the untreated control cells. (B) Relative <t>IFN-β-promoter</t> activity was assessed as in Fig. 1 after the cells were treated with increasing concentrations of γ-PGA for 30 h. (C) Schematic diagram of the SARS coronavirus replicon (top panel), which expresses a luciferase reporter gene used for cell-based analysis of the antiviral activity of γ-PGA. The Feo gene (a firefly luciferase gene fused to a neomycin phosphotransferase gene) is expressed from subgenomic mRNA synthesized from transcription-regulating sequence 9 (TRS9), allowing quantitative evaluation of viral genome replication. HEK293hTLR4 cells were transfected with plasmids expressing MD2 and CD14 along with pSARS-REP-Feo producing the SARS-CoV replicon RNA, and <t>pRL-TK.</t> At 12 h post-transfection, cells were treated with 100 μ m γ-PGA with the indicated molecular weight for 30 h and harvested for dual-luciferase assays. Firefly luciferase activity from the replicon plasmid was normalized to Renilla luciferase activity from the pRL-TK plasmid. The normalized luciferase activity of the mock-treated transfected cells was defined as 100. Data from one of two independent experiments with similar results are shown.
    Prl Tk Reporter, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Addgene inc prl tk vectors
    Inhibition by γ-PGA of SARS-CoV replicon replication. (A) The cytotoxicity of γ-PGA (MW: 2000 kDa) in HEK293hTLR4 cells transiently expressing MD2 and CD14 was evaluated by the MTS assay. Cells were treated with increasing concentrations of γ-PGA for 30 h. Data are expressed as the percentage of the untreated control cells. (B) Relative <t>IFN-β-promoter</t> activity was assessed as in Fig. 1 after the cells were treated with increasing concentrations of γ-PGA for 30 h. (C) Schematic diagram of the SARS coronavirus replicon (top panel), which expresses a luciferase reporter gene used for cell-based analysis of the antiviral activity of γ-PGA. The Feo gene (a firefly luciferase gene fused to a neomycin phosphotransferase gene) is expressed from subgenomic mRNA synthesized from transcription-regulating sequence 9 (TRS9), allowing quantitative evaluation of viral genome replication. HEK293hTLR4 cells were transfected with plasmids expressing MD2 and CD14 along with pSARS-REP-Feo producing the SARS-CoV replicon RNA, and <t>pRL-TK.</t> At 12 h post-transfection, cells were treated with 100 μ m γ-PGA with the indicated molecular weight for 30 h and harvested for dual-luciferase assays. Firefly luciferase activity from the replicon plasmid was normalized to Renilla luciferase activity from the pRL-TK plasmid. The normalized luciferase activity of the mock-treated transfected cells was defined as 100. Data from one of two independent experiments with similar results are shown.
    Prl Tk Vectors, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega standard prl tk
    Inhibition by γ-PGA of SARS-CoV replicon replication. (A) The cytotoxicity of γ-PGA (MW: 2000 kDa) in HEK293hTLR4 cells transiently expressing MD2 and CD14 was evaluated by the MTS assay. Cells were treated with increasing concentrations of γ-PGA for 30 h. Data are expressed as the percentage of the untreated control cells. (B) Relative <t>IFN-β-promoter</t> activity was assessed as in Fig. 1 after the cells were treated with increasing concentrations of γ-PGA for 30 h. (C) Schematic diagram of the SARS coronavirus replicon (top panel), which expresses a luciferase reporter gene used for cell-based analysis of the antiviral activity of γ-PGA. The Feo gene (a firefly luciferase gene fused to a neomycin phosphotransferase gene) is expressed from subgenomic mRNA synthesized from transcription-regulating sequence 9 (TRS9), allowing quantitative evaluation of viral genome replication. HEK293hTLR4 cells were transfected with plasmids expressing MD2 and CD14 along with pSARS-REP-Feo producing the SARS-CoV replicon RNA, and <t>pRL-TK.</t> At 12 h post-transfection, cells were treated with 100 μ m γ-PGA with the indicated molecular weight for 30 h and harvested for dual-luciferase assays. Firefly luciferase activity from the replicon plasmid was normalized to Renilla luciferase activity from the pRL-TK plasmid. The normalized luciferase activity of the mock-treated transfected cells was defined as 100. Data from one of two independent experiments with similar results are shown.
    Standard Prl Tk, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TaKaRa prl tk carrier
    Inhibition by γ-PGA of SARS-CoV replicon replication. (A) The cytotoxicity of γ-PGA (MW: 2000 kDa) in HEK293hTLR4 cells transiently expressing MD2 and CD14 was evaluated by the MTS assay. Cells were treated with increasing concentrations of γ-PGA for 30 h. Data are expressed as the percentage of the untreated control cells. (B) Relative <t>IFN-β-promoter</t> activity was assessed as in Fig. 1 after the cells were treated with increasing concentrations of γ-PGA for 30 h. (C) Schematic diagram of the SARS coronavirus replicon (top panel), which expresses a luciferase reporter gene used for cell-based analysis of the antiviral activity of γ-PGA. The Feo gene (a firefly luciferase gene fused to a neomycin phosphotransferase gene) is expressed from subgenomic mRNA synthesized from transcription-regulating sequence 9 (TRS9), allowing quantitative evaluation of viral genome replication. HEK293hTLR4 cells were transfected with plasmids expressing MD2 and CD14 along with pSARS-REP-Feo producing the SARS-CoV replicon RNA, and <t>pRL-TK.</t> At 12 h post-transfection, cells were treated with 100 μ m γ-PGA with the indicated molecular weight for 30 h and harvested for dual-luciferase assays. Firefly luciferase activity from the replicon plasmid was normalized to Renilla luciferase activity from the pRL-TK plasmid. The normalized luciferase activity of the mock-treated transfected cells was defined as 100. Data from one of two independent experiments with similar results are shown.
    Prl Tk Carrier, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Promega vector prl tk
    Reciprocal negative regulation of miR-130a and <t>HOTAIR.</t> (A, B) Effect of miR-130a on HOTAIR expression. GBC-SD cells were transfected with vector, miR-130a mimic (A) or miR-130a inhibitor (B) , and total RNA was isolated for qRT-PCR 24 h after transfection. (C) Schematic representation of the constructs generated for luciferase assays. (D) HOTAIR WT or its mutant devoid of specific miR-130a-binding sites in which seed matches were mutagenized from ‘TTGTAACGTGA’ to ‘GCGCCUCUUC’ was cloned downstream of Renilla luciferase gene (RLuc) in the vector <t>pRL-TK</t> and transfected into 293 T cells together with specific miRNAs mimics or the negative control mimic (NC). Luciferase assay was performed as described in Materials and Methods. Plotted are results from three independent experiments. Error bars represent S.E.M., n = 3. *p
    Vector Prl Tk, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 384 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega endogenous prl tk promoter
    Reciprocal negative regulation of miR-130a and <t>HOTAIR.</t> (A, B) Effect of miR-130a on HOTAIR expression. GBC-SD cells were transfected with vector, miR-130a mimic (A) or miR-130a inhibitor (B) , and total RNA was isolated for qRT-PCR 24 h after transfection. (C) Schematic representation of the constructs generated for luciferase assays. (D) HOTAIR WT or its mutant devoid of specific miR-130a-binding sites in which seed matches were mutagenized from ‘TTGTAACGTGA’ to ‘GCGCCUCUUC’ was cloned downstream of Renilla luciferase gene (RLuc) in the vector <t>pRL-TK</t> and transfected into 293 T cells together with specific miRNAs mimics or the negative control mimic (NC). Luciferase assay was performed as described in Materials and Methods. Plotted are results from three independent experiments. Error bars represent S.E.M., n = 3. *p
    Endogenous Prl Tk Promoter, 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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    89
    Promega prl tk luc
    The dsRNA binding domain of the NS1 protein is sufficient to prevent NF-κB activation and IFN-β induction. (A) 293 cells were transfected with a plasmid containing a reporter gene under the control of an NF-κB-responsive promoter, <t>pκB-Luc.</t> In addition, cells were cotransfected with plasmids expressing NS1, NS1(1–73), NS1(1–73,R38A/K41A), or empty vector. One day posttransfection, cells were transfected with 10 μg of dsRNA or infected with PR8, delNS1, or Sendai viruses at an MOI of 1. Two days posttransfection, luciferase activity was determined. In all transfections, <t>pRL-TK-Luc,</t> encoding a Renilla luciferase under the control of a constitutive promoter, was cotransfected, and Renilla luciferase activity was used as an internal control to normalize the results. (B) 293 cells were cotransfected with pIFN-CAT and plasmids expressing NS1, NS1(1–73), or NS1(1–73,R38A/K41A) proteins or empty vector. One day posttransfection, cells were transfected with 10 μg of dsRNA or infected with PR8 or delNS1 viruses at an MOI of 1 or infected with Sendai virus at an MOI of 10. Two days posttransfection, CAT assays were performed, and the results were quantified. (C) 293 cells were transfected with plasmids expressing NS1 or NS1(1–73) proteins or empty vector. One day posttransfection, cells were infected with delNS1 or Sendai viruses at an MOI of 1 for the indicated time points. RNA was extracted and subjected to Northern blot analysis with probes specific for IFN-β and β-actin mRNAs.
    Prl Tk Luc, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TPX blocked MAPK phosphorylation and inhibited NF‐κB activity in LPS‐treated RAW264.7 macrophages. Cells were treated with TPX in the absence or presence of LPS (1 μg · mL −1 ) for the indicated time. (A) The protein levels of phospho‐ERK, ERK, phospho‐p38, p38, phospho‐JNK and JNK were detected by Western blot analyses ( n = 6). α‐Tubulin was used as internal loading control. Data were normalized to the mean value of the LPS group. (B) The protein levels of phospho‐IKKα/β, IKKα, IKKβ, phospho‐IκBα, IκBα, phospho‐p65 and p65 were detected by Western blot analyses ( n = 6). α‐Tubulin was used as internal loading control. Data were normalized to the mean value of the LPS group. (C) Cells were pretreated with or without TPX (20 μM) for 1 h and then exposed to LPS (1 μg · mL −1 ) for another 1 h. The immunostaining of p65 was detected. Nuclei were stained with DAPI. (D) The percentage of cells undergoing p65 nuclear translocation was quantified ( n = 6). (E) The protein level of p65 in nuclear and cytoplasmic fractions was determined by Western blotting. Histone 3 and α‐tubulin were used as internal loading controls. (F) The relative expression of p65 in the nuclear fraction was quantified ( n = 6). Data are normalized to the mean value of LPS group. (G) RAW264.7 cells were co‐transfected with a NF‐kB luciferase reporter gene and pRL Renilla luciferase control reporter vector, then the cells were pretreated with TPX for 1 h followed by activation with LPS for 1 h. The transcriptional activity was expressed as the ratio of firefly to Renilla luciferase intensities in the cell lysates ( n = 6). Data are expressed as means ± SEM. # P

    Journal: British Journal of Pharmacology

    Article Title: 1,3,6,7‐Tetrahydroxy‐8‐prenylxanthone ameliorates inflammatory responses resulting from the paracrine interaction of adipocytes and macrophages) 1,3,6,7‐Tetrahydroxy‐8‐prenylxanthone ameliorates inflammatory responses resulting from the paracrine interaction of adipocytes and macrophages

    doi: 10.1111/bph.14162

    Figure Lengend Snippet: TPX blocked MAPK phosphorylation and inhibited NF‐κB activity in LPS‐treated RAW264.7 macrophages. Cells were treated with TPX in the absence or presence of LPS (1 μg · mL −1 ) for the indicated time. (A) The protein levels of phospho‐ERK, ERK, phospho‐p38, p38, phospho‐JNK and JNK were detected by Western blot analyses ( n = 6). α‐Tubulin was used as internal loading control. Data were normalized to the mean value of the LPS group. (B) The protein levels of phospho‐IKKα/β, IKKα, IKKβ, phospho‐IκBα, IκBα, phospho‐p65 and p65 were detected by Western blot analyses ( n = 6). α‐Tubulin was used as internal loading control. Data were normalized to the mean value of the LPS group. (C) Cells were pretreated with or without TPX (20 μM) for 1 h and then exposed to LPS (1 μg · mL −1 ) for another 1 h. The immunostaining of p65 was detected. Nuclei were stained with DAPI. (D) The percentage of cells undergoing p65 nuclear translocation was quantified ( n = 6). (E) The protein level of p65 in nuclear and cytoplasmic fractions was determined by Western blotting. Histone 3 and α‐tubulin were used as internal loading controls. (F) The relative expression of p65 in the nuclear fraction was quantified ( n = 6). Data are normalized to the mean value of LPS group. (G) RAW264.7 cells were co‐transfected with a NF‐kB luciferase reporter gene and pRL Renilla luciferase control reporter vector, then the cells were pretreated with TPX for 1 h followed by activation with LPS for 1 h. The transcriptional activity was expressed as the ratio of firefly to Renilla luciferase intensities in the cell lysates ( n = 6). Data are expressed as means ± SEM. # P

    Article Snippet: Plasmids of pNF‐κB‐luc and pRL‐TK were purchased from Addgene (Cambridge, MA, USA).

    Techniques: Activity Assay, Western Blot, Immunostaining, Staining, Translocation Assay, Expressing, Transfection, Luciferase, Plasmid Preparation, Activation Assay

    Inhibitory effects of G0S2 domains in different cell contexts. Transient transfection of G0S2 decreased the luciferase activity of the co-transfected PGL3E (firefly, left panel) and pRL-TK (renilla, right panel) reporter plasmids in (A) A549 cells and independently in (B) ED-1 cells. Transient transfection of G0S2 that lacked the HD domain (ΔHD) exhibited significantly less (P

    Journal: International Journal of Oncology

    Article Title: Repression of exogenous gene expression by the retinoic acid target gene G0S2

    doi: 10.3892/ijo.2013.1876

    Figure Lengend Snippet: Inhibitory effects of G0S2 domains in different cell contexts. Transient transfection of G0S2 decreased the luciferase activity of the co-transfected PGL3E (firefly, left panel) and pRL-TK (renilla, right panel) reporter plasmids in (A) A549 cells and independently in (B) ED-1 cells. Transient transfection of G0S2 that lacked the HD domain (ΔHD) exhibited significantly less (P

    Article Snippet: Plasmids and siRNAs For luciferase assay experiments, pRL-TK (Promega, Madison, WI), pGL3E (Promega), βRARE-TK-luc , pGL3-UBE1L-TK-luc ( ) and pGL3-G0S2-FL-luc ( ) were respectively used as reporter constructs.

    Techniques: Transfection, Luciferase, Activity Assay

    G0S2 inhibitory effects of G0S2 in retinoid differentiation or growth responsive cell contexts. (A) Transient transfection of G0S2 inhibited luciferase activity of retinoid responsive reporter plasmids βRARE-Tk-Luc, pGL3-UBE1L-Luc or pGL3-G0S2-FL-Luc in BEAS-2B cells, both in the presence and absence of RA (1 μ M) treatment. (B) Individual transient transfections in NT2/D1 cells (+/− RA-treatment, 1 μ M) of G0S2 inhibited activity of the reporter plasmid βRARE-Tk-Luc, which contained a retinoid responsive element, as well as the reporter plasmids pGL3E (firefly) and pRL-TK (renilla) that did not contain this responsive element. Representative results are shown from three independent experiments (each performed in triplicate) with error bars representing standard deviations. ** P

    Journal: International Journal of Oncology

    Article Title: Repression of exogenous gene expression by the retinoic acid target gene G0S2

    doi: 10.3892/ijo.2013.1876

    Figure Lengend Snippet: G0S2 inhibitory effects of G0S2 in retinoid differentiation or growth responsive cell contexts. (A) Transient transfection of G0S2 inhibited luciferase activity of retinoid responsive reporter plasmids βRARE-Tk-Luc, pGL3-UBE1L-Luc or pGL3-G0S2-FL-Luc in BEAS-2B cells, both in the presence and absence of RA (1 μ M) treatment. (B) Individual transient transfections in NT2/D1 cells (+/− RA-treatment, 1 μ M) of G0S2 inhibited activity of the reporter plasmid βRARE-Tk-Luc, which contained a retinoid responsive element, as well as the reporter plasmids pGL3E (firefly) and pRL-TK (renilla) that did not contain this responsive element. Representative results are shown from three independent experiments (each performed in triplicate) with error bars representing standard deviations. ** P

    Article Snippet: Plasmids and siRNAs For luciferase assay experiments, pRL-TK (Promega, Madison, WI), pGL3E (Promega), βRARE-TK-luc , pGL3-UBE1L-TK-luc ( ) and pGL3-G0S2-FL-luc ( ) were respectively used as reporter constructs.

    Techniques: Transfection, Luciferase, Activity Assay, Plasmid Preparation

    FXR represses fibrosis in HKC cells via downregulating Smad3 expression. ( a , b ) After treatment with TGF-β1 (2 μg/ml) for 24 h, HKC cells were treated with different concentrations of GW4064 or DMSO for another 24 h. Then the expression of Smad3, FXR, Smad2 and Smad4 were assayed by qRT-PCR ( a ) and the protein level of Smad3 was examined by Western blot ( b ). ( c , d ) After treatment with TGF-β1 (2 μg/ml) for 24 h, HKC cells were treated with FXR antagonist GS for 6 h. Then the cells were treated with GW4064 for another 24 h. The expression of Smad3 and FN were determined by qRT-PCR ( c ) and Western blot ( d ). ( e ) HKC cells were transiently cotransfected with the luciferase reporter pGL3-Smad3 containing Smad3 promoter region and the renilla luciferase expression vector pRL-TK for 6 h, followed by replacing transfection medium with complete medium and incubating for 12 h. Then the cells were treated with GW4064 (5 μM) or DMSO for 24 h. Subsequently, the cells were collected and luciferase activities were measured using dual-luciferase assay kit. The firefly luciferase activity was normalized with renilla luciferase activity. Data are Mean ± SD from 3 assays in triplicates. ( f ) HKC cells were transiently transfected with Smad3 expression vector pcDNA3.1-Smad3 for 6 h, followed by replacing the transfection medium with complete medium and incubating for 12 h. Then the cells were treated with TGF-β1 (2 μg/ml) for 24 h, followed by the treatment with GW4064 (5 μM) or vehicle DMSO for another 24 h. Thereafter, the protein levels of Smad3 and FN were determined by Western blot. * P

    Journal: Scientific Reports

    Article Title: Activation of FXR protects against renal fibrosis via suppressing Smad3 expression

    doi: 10.1038/srep37234

    Figure Lengend Snippet: FXR represses fibrosis in HKC cells via downregulating Smad3 expression. ( a , b ) After treatment with TGF-β1 (2 μg/ml) for 24 h, HKC cells were treated with different concentrations of GW4064 or DMSO for another 24 h. Then the expression of Smad3, FXR, Smad2 and Smad4 were assayed by qRT-PCR ( a ) and the protein level of Smad3 was examined by Western blot ( b ). ( c , d ) After treatment with TGF-β1 (2 μg/ml) for 24 h, HKC cells were treated with FXR antagonist GS for 6 h. Then the cells were treated with GW4064 for another 24 h. The expression of Smad3 and FN were determined by qRT-PCR ( c ) and Western blot ( d ). ( e ) HKC cells were transiently cotransfected with the luciferase reporter pGL3-Smad3 containing Smad3 promoter region and the renilla luciferase expression vector pRL-TK for 6 h, followed by replacing transfection medium with complete medium and incubating for 12 h. Then the cells were treated with GW4064 (5 μM) or DMSO for 24 h. Subsequently, the cells were collected and luciferase activities were measured using dual-luciferase assay kit. The firefly luciferase activity was normalized with renilla luciferase activity. Data are Mean ± SD from 3 assays in triplicates. ( f ) HKC cells were transiently transfected with Smad3 expression vector pcDNA3.1-Smad3 for 6 h, followed by replacing the transfection medium with complete medium and incubating for 12 h. Then the cells were treated with TGF-β1 (2 μg/ml) for 24 h, followed by the treatment with GW4064 (5 μM) or vehicle DMSO for another 24 h. Thereafter, the protein levels of Smad3 and FN were determined by Western blot. * P

    Article Snippet: Oligo (dT) primer, Dual-luciferase assay kits, pGL3-basic and pRL-TK vectors were from Promega (Madison, WI, USA).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Plasmid Preparation, Transfection, Activity Assay

    I14 in mature LEP is essential for LEP-LEPR interaction and the downstream STAT3 pathway. ( A ) Sanger-sequencing of the RT-PCR products showed that the Lep mRNA of Lep ∆ I14/ ∆ I14 rat had a deletion of 3 nucleotides ATC encoding an Ile residue. ( B ) Western blot showed that the mature LEP ∆I14 protein is stably expressed in the WAT of Lep ∆ I14/ ∆ I14 rats. Shown is one of three independent experiments. ( C ) ELISA showed that serum LEP ∆I14 in male Lep ∆ I14/ ∆ I14 rats (n = 5) is significantly increased compared to that of serum LEP WT in the male WT controls (n = 5). ( D ) Computer assimilation of LEP-LEPR interaction using available information from their human homologs: LEP (PDB number: 1AX8) and LEPR (PDB number: 3V6O). ( E ) STAT3 reporter assay. 293FT cells were treated with WT and mutant recombinant rat LEP proteins at different concentrations after transient transfection of pcDNA-Lepr, pRL-TK and pGL6-Stat3. Relative luciferase activity was determined by firefly luciferase light units normalized by that of renilla luciferase.

    Journal: Scientific Reports

    Article Title: The 14th Ile residue is essential for Leptin function in regulating energy homeostasis in rat

    doi: 10.1038/srep28508

    Figure Lengend Snippet: I14 in mature LEP is essential for LEP-LEPR interaction and the downstream STAT3 pathway. ( A ) Sanger-sequencing of the RT-PCR products showed that the Lep mRNA of Lep ∆ I14/ ∆ I14 rat had a deletion of 3 nucleotides ATC encoding an Ile residue. ( B ) Western blot showed that the mature LEP ∆I14 protein is stably expressed in the WAT of Lep ∆ I14/ ∆ I14 rats. Shown is one of three independent experiments. ( C ) ELISA showed that serum LEP ∆I14 in male Lep ∆ I14/ ∆ I14 rats (n = 5) is significantly increased compared to that of serum LEP WT in the male WT controls (n = 5). ( D ) Computer assimilation of LEP-LEPR interaction using available information from their human homologs: LEP (PDB number: 1AX8) and LEPR (PDB number: 3V6O). ( E ) STAT3 reporter assay. 293FT cells were treated with WT and mutant recombinant rat LEP proteins at different concentrations after transient transfection of pcDNA-Lepr, pRL-TK and pGL6-Stat3. Relative luciferase activity was determined by firefly luciferase light units normalized by that of renilla luciferase.

    Article Snippet: The cells were seeded in a 96-well plate (Corning) and transfected with the human LEPR overexpression vector pcDNA-Lepr, the control pRL-TK (Promega) and the pGL6-Stat3 plasmid which contains the luciferase gene under control of the STAT3-inducible promoter.

    Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Stable Transfection, Enzyme-linked Immunosorbent Assay, Reporter Assay, Mutagenesis, Recombinant, Transfection, Luciferase, Activity Assay

    FoxO1 directly bound to the FRE site in the FoxO1 promoter. ( a ) A 2048-bp fragment of the FoxO1 promoter was amplified with PCR using mouse genomic DNA (bases 78–2125, 5'-UTR of FoxO1 ) containing a FRE (AGTAAACAAA) and cloned into the pGL3-Basic plasmid as pGL3-FoxO1 (W). pGL3-FoxO1 (M) was obtained by introducing four mutations (italics) into the FRE site. ( b ) FoxO1 reporter activities in mouse 3T3 cells co-transfected with FoxO1 expression vectors and FoxO1 promoter constructs for 36 h. The reporter activities were normalized to those of pRL-TK. RLA, relative luciferase activities. ( c – e ) The binding of FoxO1 to the FoxO1 promoter in MGCs or 3T3 fibroblasts was detected with ChIP assays following FSH treatment for 6 h. DNA was isolated from the precipitated complexes as a template for qRT-PCR ( c ). The qRT-PCR products were then analyzed on a 2% agarose gel ( d ) and quantified with densitometry using ImageJ 1.42q software ( e ). The amount of immunoprecipitated DNA for each ChIP reaction is shown as the percentage of input chromatin. IgG was the negative control. The data represent the means±S.E. ( n =3). * P

    Journal: Cell Death & Disease

    Article Title: Involvement of FoxO1 in the effects of follicle-stimulating hormone on inhibition of apoptosis in mouse granulosa cells

    doi: 10.1038/cddis.2014.400

    Figure Lengend Snippet: FoxO1 directly bound to the FRE site in the FoxO1 promoter. ( a ) A 2048-bp fragment of the FoxO1 promoter was amplified with PCR using mouse genomic DNA (bases 78–2125, 5'-UTR of FoxO1 ) containing a FRE (AGTAAACAAA) and cloned into the pGL3-Basic plasmid as pGL3-FoxO1 (W). pGL3-FoxO1 (M) was obtained by introducing four mutations (italics) into the FRE site. ( b ) FoxO1 reporter activities in mouse 3T3 cells co-transfected with FoxO1 expression vectors and FoxO1 promoter constructs for 36 h. The reporter activities were normalized to those of pRL-TK. RLA, relative luciferase activities. ( c – e ) The binding of FoxO1 to the FoxO1 promoter in MGCs or 3T3 fibroblasts was detected with ChIP assays following FSH treatment for 6 h. DNA was isolated from the precipitated complexes as a template for qRT-PCR ( c ). The qRT-PCR products were then analyzed on a 2% agarose gel ( d ) and quantified with densitometry using ImageJ 1.42q software ( e ). The amount of immunoprecipitated DNA for each ChIP reaction is shown as the percentage of input chromatin. IgG was the negative control. The data represent the means±S.E. ( n =3). * P

    Article Snippet: Three plasmids were co-transfected in each treatment: (1) 1.6 μ g expression vector pcDNA3-FLAG-FKHR (WT); (2) 1.6 μ g reporter construct pGL3-FoxO1 (W) or pGL3-FoxO1 (M); and (3) 32 ng control reporter pRL-TK (Promega).

    Techniques: Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Transfection, Expressing, Construct, Luciferase, Binding Assay, Chromatin Immunoprecipitation, Isolation, Quantitative RT-PCR, Agarose Gel Electrophoresis, Software, Immunoprecipitation, Negative Control

    TAK1 inhibition of HBV replication and gene expression requires activation of downstream MAPK pathways. ( A ) Huh7 cells were co-transfected with 1.5 μg of pHBV1.3 and 1.5 μg of pXJ40-HA, or plasmids expressing HA-hTAK1, DN-hTAK1 or CA-hTAK1. Cells were harvested 72 h after transfection. HBV RI, viral RNAs and HBsAg and HBeAg were determined as described above. HA-tag and β-actin (loading control) were examined by Western blot, 28 S/18 S rRNAs served as loading controls for Northern blot analysis. ( B ) Huh7 cells were co-transfected with 100 ng of pCP or pXP, and 100 ng of pXJ40-HA or plasmids expressing HA-hTAK1, DN-TAK1 or CA-TAK1. Cells were harvested 48 h after transfection and firefly and Renilla luciferase activities were measured and normalized against those generated by the pGL3-basic control plasmid. ( C ) Huh7 cells were transfected with 1.5 μg of pXJ40-HA or plasmids expressing HA-hTAK1, DN-TAK1, and CA-TAK1. Phosphorylation and basal expression levels of p38, JNK, ERK, and NF-κBp 65 were analyzed by Western blot 12 h after transfection, using β-actin as loading control. ( D ) Huh7 cells were seeded in 24-well plates and transfected with 100 ng of the reporter plasmids pNF-κB-luc (pNF-kB) or pAP1-luc (pAP-1) and 100 ng of pXJ40-HA, hTAK1, DN-TAK1, or CA-TAK1. For each transfection, 100 ng of pRL-TK was included as an internal control of transfection efficiency. Cells were harvested 48 h after transfection and firefly and Renilla luciferase activities were measured and normalized against those generated by the pGL3-basic control plasmid.

    Journal: Scientific Reports

    Article Title: Transforming growth factor β-activated kinase 1 transcriptionally suppresses hepatitis B virus replication

    doi: 10.1038/srep39901

    Figure Lengend Snippet: TAK1 inhibition of HBV replication and gene expression requires activation of downstream MAPK pathways. ( A ) Huh7 cells were co-transfected with 1.5 μg of pHBV1.3 and 1.5 μg of pXJ40-HA, or plasmids expressing HA-hTAK1, DN-hTAK1 or CA-hTAK1. Cells were harvested 72 h after transfection. HBV RI, viral RNAs and HBsAg and HBeAg were determined as described above. HA-tag and β-actin (loading control) were examined by Western blot, 28 S/18 S rRNAs served as loading controls for Northern blot analysis. ( B ) Huh7 cells were co-transfected with 100 ng of pCP or pXP, and 100 ng of pXJ40-HA or plasmids expressing HA-hTAK1, DN-TAK1 or CA-TAK1. Cells were harvested 48 h after transfection and firefly and Renilla luciferase activities were measured and normalized against those generated by the pGL3-basic control plasmid. ( C ) Huh7 cells were transfected with 1.5 μg of pXJ40-HA or plasmids expressing HA-hTAK1, DN-TAK1, and CA-TAK1. Phosphorylation and basal expression levels of p38, JNK, ERK, and NF-κBp 65 were analyzed by Western blot 12 h after transfection, using β-actin as loading control. ( D ) Huh7 cells were seeded in 24-well plates and transfected with 100 ng of the reporter plasmids pNF-κB-luc (pNF-kB) or pAP1-luc (pAP-1) and 100 ng of pXJ40-HA, hTAK1, DN-TAK1, or CA-TAK1. For each transfection, 100 ng of pRL-TK was included as an internal control of transfection efficiency. Cells were harvested 48 h after transfection and firefly and Renilla luciferase activities were measured and normalized against those generated by the pGL3-basic control plasmid.

    Article Snippet: Luciferase reporter vectors pNF-κB-luc, pAP-1-luc, and pRL-TK were purchased from Clontech (Mountain View, CA, USA).

    Techniques: Inhibition, Expressing, Activation Assay, Transfection, Western Blot, Northern Blot, Luciferase, Generated, Plasmid Preparation

    Activity-induced reporter gene expression in N2A cells. For KC1 stimulation experiments, tetR-VP16-GS-hNFAT was co-expressed with tetO-Fluc and pRL-TK. When stimulated with 60 mM KC1, the FLuc/RLuc ratio increased nearly 2-fold. For light stimulation experiments, Gal4-NFATAD was co-expressed with VChR1, UAS-Fluc (pFR-Luc), and pRL-TK. After 488-nm light illumination for 12 hours, the Fluc/Rluc ratio increased by about 5-fold. Each data point represents average results from two samples.

    Journal: Journal of Neurogenetics

    Article Title: Mapping Neural Circuits with Activity-Dependent Nuclear Import of a Transcription Factor

    doi: 10.3109/01677063.2011.642910

    Figure Lengend Snippet: Activity-induced reporter gene expression in N2A cells. For KC1 stimulation experiments, tetR-VP16-GS-hNFAT was co-expressed with tetO-Fluc and pRL-TK. When stimulated with 60 mM KC1, the FLuc/RLuc ratio increased nearly 2-fold. For light stimulation experiments, Gal4-NFATAD was co-expressed with VChR1, UAS-Fluc (pFR-Luc), and pRL-TK. After 488-nm light illumination for 12 hours, the Fluc/Rluc ratio increased by about 5-fold. Each data point represents average results from two samples.

    Article Snippet: Lipofectamine 2000 (Invitrogen, xx, xx) was used to transfect N2A cells with tetR-VP16-GS-hNFATc1, tetO-Fluc, and pRL-TK (Stratagene, xx, xx) at a ratio of 1:0.2:0.1.

    Techniques: Activity Assay, Expressing

    Regions both 5′ and 3′ to the start of transcription were required for SF-1 promoter activity. A region of the Ftz-F1 gene from −734 to +60 bp, relative to the SF-1 transcriptional start site (+1), was cloned upstream of the firefly luciferase reporter gene. Promoter activity was determined by transient transfection analysis in either primary rat Sertoli cells, MSC-1 cells, or MA-10 cells as described in Material and Methods. Promoter constructs were cotransfected with pRL-TK, which expresses Renilla luciferase from the thymidine kinase promoter, and was used to control for transfection efficiency. In both A and B, firefly luciferase activity from the different promoter constructs was normalized to Renilla luciferase values. In A, the firefly/ Renilla luciferase activity of the SF-1 (−734/+60) construct was assayed in each of the cell lines and reported as the activity relative to the firefly/ Renilla luciferase activity of the promoterless control vector pGL3-Basic. In B, various promoter deletion mutants are indicated; the activity of each, determined as described above, was analyzed in each of the cell types. Here, the data represent the firefly/ Renilla luciferase activity of each SF-1 promoter made relative to the firefly/ Renilla luciferase activity of the SF-1 (−734/+60) Luc construct. Transfections were done a minimum of three times. Error bars represent the SEM.

    Journal: Biology of reproduction

    Article Title: Expression of Steroidogenic Factor 1 in the Testis Requires an E Box and CCAAT Box in its Promoter Proximal Region 1

    doi:

    Figure Lengend Snippet: Regions both 5′ and 3′ to the start of transcription were required for SF-1 promoter activity. A region of the Ftz-F1 gene from −734 to +60 bp, relative to the SF-1 transcriptional start site (+1), was cloned upstream of the firefly luciferase reporter gene. Promoter activity was determined by transient transfection analysis in either primary rat Sertoli cells, MSC-1 cells, or MA-10 cells as described in Material and Methods. Promoter constructs were cotransfected with pRL-TK, which expresses Renilla luciferase from the thymidine kinase promoter, and was used to control for transfection efficiency. In both A and B, firefly luciferase activity from the different promoter constructs was normalized to Renilla luciferase values. In A, the firefly/ Renilla luciferase activity of the SF-1 (−734/+60) construct was assayed in each of the cell lines and reported as the activity relative to the firefly/ Renilla luciferase activity of the promoterless control vector pGL3-Basic. In B, various promoter deletion mutants are indicated; the activity of each, determined as described above, was analyzed in each of the cell types. Here, the data represent the firefly/ Renilla luciferase activity of each SF-1 promoter made relative to the firefly/ Renilla luciferase activity of the SF-1 (−734/+60) Luc construct. Transfections were done a minimum of three times. Error bars represent the SEM.

    Article Snippet: Four to six hours later, cells were transfected using 0.2 μg of pGL3-reporter vector and 45 ng of pRL-TK in combination with 1 μl lipofectamine and 6 μl Plus reagent according to the manufacturer’s recommendations (Life Technologies); pRL-TK expresses Renilla luciferase from the herpes simplex virus thymidine kinase promoter and was included to control for transfection efficiency.

    Techniques: Activity Assay, Clone Assay, Luciferase, Transfection, Construct, Plasmid Preparation

    Inhibition by γ-PGA of SARS-CoV replicon replication. (A) The cytotoxicity of γ-PGA (MW: 2000 kDa) in HEK293hTLR4 cells transiently expressing MD2 and CD14 was evaluated by the MTS assay. Cells were treated with increasing concentrations of γ-PGA for 30 h. Data are expressed as the percentage of the untreated control cells. (B) Relative IFN-β-promoter activity was assessed as in Fig. 1 after the cells were treated with increasing concentrations of γ-PGA for 30 h. (C) Schematic diagram of the SARS coronavirus replicon (top panel), which expresses a luciferase reporter gene used for cell-based analysis of the antiviral activity of γ-PGA. The Feo gene (a firefly luciferase gene fused to a neomycin phosphotransferase gene) is expressed from subgenomic mRNA synthesized from transcription-regulating sequence 9 (TRS9), allowing quantitative evaluation of viral genome replication. HEK293hTLR4 cells were transfected with plasmids expressing MD2 and CD14 along with pSARS-REP-Feo producing the SARS-CoV replicon RNA, and pRL-TK. At 12 h post-transfection, cells were treated with 100 μ m γ-PGA with the indicated molecular weight for 30 h and harvested for dual-luciferase assays. Firefly luciferase activity from the replicon plasmid was normalized to Renilla luciferase activity from the pRL-TK plasmid. The normalized luciferase activity of the mock-treated transfected cells was defined as 100. Data from one of two independent experiments with similar results are shown.

    Journal: Biomaterials

    Article Title: The antiviral activity of poly-γ-glutamic acid, a polypeptide secreted by Bacillus sp., through induction of CD14-dependent type I interferon responses

    doi: 10.1016/j.biomaterials.2013.08.067

    Figure Lengend Snippet: Inhibition by γ-PGA of SARS-CoV replicon replication. (A) The cytotoxicity of γ-PGA (MW: 2000 kDa) in HEK293hTLR4 cells transiently expressing MD2 and CD14 was evaluated by the MTS assay. Cells were treated with increasing concentrations of γ-PGA for 30 h. Data are expressed as the percentage of the untreated control cells. (B) Relative IFN-β-promoter activity was assessed as in Fig. 1 after the cells were treated with increasing concentrations of γ-PGA for 30 h. (C) Schematic diagram of the SARS coronavirus replicon (top panel), which expresses a luciferase reporter gene used for cell-based analysis of the antiviral activity of γ-PGA. The Feo gene (a firefly luciferase gene fused to a neomycin phosphotransferase gene) is expressed from subgenomic mRNA synthesized from transcription-regulating sequence 9 (TRS9), allowing quantitative evaluation of viral genome replication. HEK293hTLR4 cells were transfected with plasmids expressing MD2 and CD14 along with pSARS-REP-Feo producing the SARS-CoV replicon RNA, and pRL-TK. At 12 h post-transfection, cells were treated with 100 μ m γ-PGA with the indicated molecular weight for 30 h and harvested for dual-luciferase assays. Firefly luciferase activity from the replicon plasmid was normalized to Renilla luciferase activity from the pRL-TK plasmid. The normalized luciferase activity of the mock-treated transfected cells was defined as 100. Data from one of two independent experiments with similar results are shown.

    Article Snippet: 2.3 Interferon β reporter assay HEK293hTLR4 cells grown in 24-well plates were transfected with the plasmid expressing either human CD14 or MD2 (150 ng each) plus 150 ng of IFNβ-pGL3, which expresses firefly luciferase under the control of the IFN-β promoter and 150 ng of pRL-TK reporter (Promega, Madison, WI, USA), which expresses Renilla luciferase as an internal control.

    Techniques: Inhibition, Expressing, MTS Assay, Activity Assay, Luciferase, Synthesized, Sequencing, Transfection, Molecular Weight, Plasmid Preparation

    Stimulation of TLR4 by γ-PGA requires the accessory factors MD2 and CD14 for induction of IFN-β production. (A) HEK293hTLR4 cells were transfected with plasmids to express the indicated proteins (MD2 and CD14) and with the luciferase expressing plasmids (IFNβ-pGL3 and pRL-TK) for the IFN-β promoter activity assay. After 12 h, the cells were treated with 100 μ m γ-PGA (MW = 2000 kDa) or 200 ng/ml LPS for 30 h and then harvested for dual luciferase assays. Firefly luciferase activity was normalized to Renilla luciferase activity from the pRL-TK plasmid. Normalized luciferase activity (Fluc/Rluc) of mock-treated cells was defined as 100. Data are presented as means ± SD of three measurements from three independent experiments. * P

    Journal: Biomaterials

    Article Title: The antiviral activity of poly-γ-glutamic acid, a polypeptide secreted by Bacillus sp., through induction of CD14-dependent type I interferon responses

    doi: 10.1016/j.biomaterials.2013.08.067

    Figure Lengend Snippet: Stimulation of TLR4 by γ-PGA requires the accessory factors MD2 and CD14 for induction of IFN-β production. (A) HEK293hTLR4 cells were transfected with plasmids to express the indicated proteins (MD2 and CD14) and with the luciferase expressing plasmids (IFNβ-pGL3 and pRL-TK) for the IFN-β promoter activity assay. After 12 h, the cells were treated with 100 μ m γ-PGA (MW = 2000 kDa) or 200 ng/ml LPS for 30 h and then harvested for dual luciferase assays. Firefly luciferase activity was normalized to Renilla luciferase activity from the pRL-TK plasmid. Normalized luciferase activity (Fluc/Rluc) of mock-treated cells was defined as 100. Data are presented as means ± SD of three measurements from three independent experiments. * P

    Article Snippet: 2.3 Interferon β reporter assay HEK293hTLR4 cells grown in 24-well plates were transfected with the plasmid expressing either human CD14 or MD2 (150 ng each) plus 150 ng of IFNβ-pGL3, which expresses firefly luciferase under the control of the IFN-β promoter and 150 ng of pRL-TK reporter (Promega, Madison, WI, USA), which expresses Renilla luciferase as an internal control.

    Techniques: Transfection, Luciferase, Expressing, Activity Assay, Plasmid Preparation

    Reciprocal negative regulation of miR-130a and HOTAIR. (A, B) Effect of miR-130a on HOTAIR expression. GBC-SD cells were transfected with vector, miR-130a mimic (A) or miR-130a inhibitor (B) , and total RNA was isolated for qRT-PCR 24 h after transfection. (C) Schematic representation of the constructs generated for luciferase assays. (D) HOTAIR WT or its mutant devoid of specific miR-130a-binding sites in which seed matches were mutagenized from ‘TTGTAACGTGA’ to ‘GCGCCUCUUC’ was cloned downstream of Renilla luciferase gene (RLuc) in the vector pRL-TK and transfected into 293 T cells together with specific miRNAs mimics or the negative control mimic (NC). Luciferase assay was performed as described in Materials and Methods. Plotted are results from three independent experiments. Error bars represent S.E.M., n = 3. *p

    Journal: Molecular Cancer

    Article Title: Long non-coding RNA HOTAIR, a c-Myc activated driver of malignancy, negatively regulates miRNA-130a in gallbladder cancer

    doi: 10.1186/1476-4598-13-156

    Figure Lengend Snippet: Reciprocal negative regulation of miR-130a and HOTAIR. (A, B) Effect of miR-130a on HOTAIR expression. GBC-SD cells were transfected with vector, miR-130a mimic (A) or miR-130a inhibitor (B) , and total RNA was isolated for qRT-PCR 24 h after transfection. (C) Schematic representation of the constructs generated for luciferase assays. (D) HOTAIR WT or its mutant devoid of specific miR-130a-binding sites in which seed matches were mutagenized from ‘TTGTAACGTGA’ to ‘GCGCCUCUUC’ was cloned downstream of Renilla luciferase gene (RLuc) in the vector pRL-TK and transfected into 293 T cells together with specific miRNAs mimics or the negative control mimic (NC). Luciferase assay was performed as described in Materials and Methods. Plotted are results from three independent experiments. Error bars represent S.E.M., n = 3. *p

    Article Snippet: For the dual luciferase activity, LncRNA-HOTAIR (lncRNA-HOTAIR-wt) or its mutant devoid of specific miRNA binding sites (lncRNA-HOTAIR-mu) was cloned into 3’UTR of the Renilla luciferase gene in the vector pRL-TK (Promega, Madison, WI, USA).

    Techniques: Expressing, Transfection, Plasmid Preparation, Isolation, Quantitative RT-PCR, Construct, Generated, Luciferase, Mutagenesis, Binding Assay, Clone Assay, Negative Control

    Fe-IRF1 is located in the nucleus and can activate the IFN- β pathway. (a) The N terminus sequences from 1 to 125 amino acid of human, mouse, rat, pig, bovine, dog, and cat IRF1 were aligned using MEGA5. (b) CRFK cells were transfected with pEGFP-IRF1 and subjected to confocal microscopy. (c, d) CRFK cells were cotransfected with 0.25 μ g/well of the reporter plasmid pIFN- β -Luc (c) or pISRE-TA-Luc (d) and 0.25 μ g/well of p3×Flag-IRF1 or empty vector, along with 0.02 μ g /well of pRL-TK plasmid. Luciferase assays were performed at 24 h posttransfection. The SeV infection group serves as a positive control. The data shown represent the mean ± SD for three independent experiments. ∗ : P

    Journal: BioMed Research International

    Article Title: Identification of Feline Interferon Regulatory Factor 1 as an Efficient Antiviral Factor against the Replication of Feline Calicivirus and Other Feline Viruses

    doi: 10.1155/2018/2739830

    Figure Lengend Snippet: Fe-IRF1 is located in the nucleus and can activate the IFN- β pathway. (a) The N terminus sequences from 1 to 125 amino acid of human, mouse, rat, pig, bovine, dog, and cat IRF1 were aligned using MEGA5. (b) CRFK cells were transfected with pEGFP-IRF1 and subjected to confocal microscopy. (c, d) CRFK cells were cotransfected with 0.25 μ g/well of the reporter plasmid pIFN- β -Luc (c) or pISRE-TA-Luc (d) and 0.25 μ g/well of p3×Flag-IRF1 or empty vector, along with 0.02 μ g /well of pRL-TK plasmid. Luciferase assays were performed at 24 h posttransfection. The SeV infection group serves as a positive control. The data shown represent the mean ± SD for three independent experiments. ∗ : P

    Article Snippet: Briefly, CRFK cells (5×104 /well) grown in 48-well plates were cotransfected with 0.5 μ g/well reporter plasmid, 0.02 μ g/well plasmid pRL-TK (Promega) (as an internal control for normalization of the assay system), or the indicated expression plasmid.

    Techniques: Transfection, Confocal Microscopy, Plasmid Preparation, Luciferase, Infection, Positive Control

    The dsRNA binding domain of the NS1 protein is sufficient to prevent NF-κB activation and IFN-β induction. (A) 293 cells were transfected with a plasmid containing a reporter gene under the control of an NF-κB-responsive promoter, pκB-Luc. In addition, cells were cotransfected with plasmids expressing NS1, NS1(1–73), NS1(1–73,R38A/K41A), or empty vector. One day posttransfection, cells were transfected with 10 μg of dsRNA or infected with PR8, delNS1, or Sendai viruses at an MOI of 1. Two days posttransfection, luciferase activity was determined. In all transfections, pRL-TK-Luc, encoding a Renilla luciferase under the control of a constitutive promoter, was cotransfected, and Renilla luciferase activity was used as an internal control to normalize the results. (B) 293 cells were cotransfected with pIFN-CAT and plasmids expressing NS1, NS1(1–73), or NS1(1–73,R38A/K41A) proteins or empty vector. One day posttransfection, cells were transfected with 10 μg of dsRNA or infected with PR8 or delNS1 viruses at an MOI of 1 or infected with Sendai virus at an MOI of 10. Two days posttransfection, CAT assays were performed, and the results were quantified. (C) 293 cells were transfected with plasmids expressing NS1 or NS1(1–73) proteins or empty vector. One day posttransfection, cells were infected with delNS1 or Sendai viruses at an MOI of 1 for the indicated time points. RNA was extracted and subjected to Northern blot analysis with probes specific for IFN-β and β-actin mRNAs.

    Journal: Journal of Virology

    Article Title: Influenza A Virus NS1 Protein Prevents Activation of NF-?B and Induction of Alpha/Beta Interferon

    doi:

    Figure Lengend Snippet: The dsRNA binding domain of the NS1 protein is sufficient to prevent NF-κB activation and IFN-β induction. (A) 293 cells were transfected with a plasmid containing a reporter gene under the control of an NF-κB-responsive promoter, pκB-Luc. In addition, cells were cotransfected with plasmids expressing NS1, NS1(1–73), NS1(1–73,R38A/K41A), or empty vector. One day posttransfection, cells were transfected with 10 μg of dsRNA or infected with PR8, delNS1, or Sendai viruses at an MOI of 1. Two days posttransfection, luciferase activity was determined. In all transfections, pRL-TK-Luc, encoding a Renilla luciferase under the control of a constitutive promoter, was cotransfected, and Renilla luciferase activity was used as an internal control to normalize the results. (B) 293 cells were cotransfected with pIFN-CAT and plasmids expressing NS1, NS1(1–73), or NS1(1–73,R38A/K41A) proteins or empty vector. One day posttransfection, cells were transfected with 10 μg of dsRNA or infected with PR8 or delNS1 viruses at an MOI of 1 or infected with Sendai virus at an MOI of 10. Two days posttransfection, CAT assays were performed, and the results were quantified. (C) 293 cells were transfected with plasmids expressing NS1 or NS1(1–73) proteins or empty vector. One day posttransfection, cells were infected with delNS1 or Sendai viruses at an MOI of 1 for the indicated time points. RNA was extracted and subjected to Northern blot analysis with probes specific for IFN-β and β-actin mRNAs.

    Article Snippet: Plasmid pκB-Luc encodes a firefly luciferase reporter gene under the control of an NF-κB-responsive promoter ( ). pRL-TK-Luc (Promega), containing a Renilla luciferase reporter gene under the control of the herpes simplex virus thymidine kinase promoter, was used as an internal control of transfection efficiency. pCAGGS-NS1(SAM) was made by inserting the ORF of the NS1 of wild-type PR8 virus between the Eco RI and Xho I sites of pCAGGS , as described previously ( ).

    Techniques: Binding Assay, Activation Assay, Transfection, Plasmid Preparation, Expressing, Infection, Luciferase, Activity Assay, Northern Blot