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In vitro characterization of wild-type and His257Arg (H257R) mutant TrpRS proteins. ( A ) β-Gal reporter assay and ( B ) firefly luciferase reporter assay demonstrating that H257R TrpRS has a dominant-negative effect on protein synthesis. <t>HEK293</t> cells co-transfected with β-Gal or firefly luciferase reporter plasmids, along with different ratios of wild-type and H257R TrpRS expression plasmids were lysed and assayed for β-Gal or firefly luciferase activities at 48 h after transfection. The error bars indicate standard errors of the mean ( n = 3) and asterisks indicate statistically significant differences (** P
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1) Product Images from "A recurrent WARS mutation is a novel cause of autosomal dominant distal hereditary motor neuropathy"

Article Title: A recurrent WARS mutation is a novel cause of autosomal dominant distal hereditary motor neuropathy

Journal: Brain

doi: 10.1093/brain/awx058

In vitro characterization of wild-type and His257Arg (H257R) mutant TrpRS proteins. ( A ) β-Gal reporter assay and ( B ) firefly luciferase reporter assay demonstrating that H257R TrpRS has a dominant-negative effect on protein synthesis. HEK293 cells co-transfected with β-Gal or firefly luciferase reporter plasmids, along with different ratios of wild-type and H257R TrpRS expression plasmids were lysed and assayed for β-Gal or firefly luciferase activities at 48 h after transfection. The error bars indicate standard errors of the mean ( n = 3) and asterisks indicate statistically significant differences (** P
Figure Legend Snippet: In vitro characterization of wild-type and His257Arg (H257R) mutant TrpRS proteins. ( A ) β-Gal reporter assay and ( B ) firefly luciferase reporter assay demonstrating that H257R TrpRS has a dominant-negative effect on protein synthesis. HEK293 cells co-transfected with β-Gal or firefly luciferase reporter plasmids, along with different ratios of wild-type and H257R TrpRS expression plasmids were lysed and assayed for β-Gal or firefly luciferase activities at 48 h after transfection. The error bars indicate standard errors of the mean ( n = 3) and asterisks indicate statistically significant differences (** P

Techniques Used: In Vitro, Mutagenesis, Reporter Assay, Luciferase, Dominant Negative Mutation, Transfection, Expressing

2) Product Images from "A recurrent WARS mutation is a novel cause of autosomal dominant distal hereditary motor neuropathy"

Article Title: A recurrent WARS mutation is a novel cause of autosomal dominant distal hereditary motor neuropathy

Journal: Brain

doi: 10.1093/brain/awx058

In vitro characterization of wild-type and His257Arg (H257R) mutant TrpRS proteins. ( A ) β-Gal reporter assay and ( B ) firefly luciferase reporter assay demonstrating that H257R TrpRS has a dominant-negative effect on protein synthesis. HEK293 cells co-transfected with β-Gal or firefly luciferase reporter plasmids, along with different ratios of wild-type and H257R TrpRS expression plasmids were lysed and assayed for β-Gal or firefly luciferase activities at 48 h after transfection. The error bars indicate standard errors of the mean ( n = 3) and asterisks indicate statistically significant differences (** P
Figure Legend Snippet: In vitro characterization of wild-type and His257Arg (H257R) mutant TrpRS proteins. ( A ) β-Gal reporter assay and ( B ) firefly luciferase reporter assay demonstrating that H257R TrpRS has a dominant-negative effect on protein synthesis. HEK293 cells co-transfected with β-Gal or firefly luciferase reporter plasmids, along with different ratios of wild-type and H257R TrpRS expression plasmids were lysed and assayed for β-Gal or firefly luciferase activities at 48 h after transfection. The error bars indicate standard errors of the mean ( n = 3) and asterisks indicate statistically significant differences (** P

Techniques Used: In Vitro, Mutagenesis, Reporter Assay, Luciferase, Dominant Negative Mutation, Transfection, Expressing

His257Arg (H257R) TrpRS inhibits neurite outgrowth and leads to neurite degeneration. Neuro-2a (N2a) ( A ) or SH-SY5Y ( B ) cells were transfected with expression vector containing wild-type (WT) or H257R TrpRS or empty vector (vector control), grown under differentiation conditions, and immunostained against Myc (exogenous TrpRS staining) and neurofilament heavy polypeptide (NFH; neurite staining) at 72 h post-transfection. The representative fluorescence-immunostaining and phase-contrast images were shown, along with statistical results of per cent of cells bearing neurites and average primary neurite length. Scale bar = 50 μm. At least 100 cells from three independent experiments were measured for each preparation and data were expressed as the mean ± standard error of the mean (SEM). * P -value
Figure Legend Snippet: His257Arg (H257R) TrpRS inhibits neurite outgrowth and leads to neurite degeneration. Neuro-2a (N2a) ( A ) or SH-SY5Y ( B ) cells were transfected with expression vector containing wild-type (WT) or H257R TrpRS or empty vector (vector control), grown under differentiation conditions, and immunostained against Myc (exogenous TrpRS staining) and neurofilament heavy polypeptide (NFH; neurite staining) at 72 h post-transfection. The representative fluorescence-immunostaining and phase-contrast images were shown, along with statistical results of per cent of cells bearing neurites and average primary neurite length. Scale bar = 50 μm. At least 100 cells from three independent experiments were measured for each preparation and data were expressed as the mean ± standard error of the mean (SEM). * P -value

Techniques Used: Transfection, Expressing, Plasmid Preparation, Staining, Fluorescence, Immunostaining

3) Product Images from "Isoflavones enhance interleukin-17 gene expression via retinoic acid receptor-related orphan receptors α and γ"

Article Title: Isoflavones enhance interleukin-17 gene expression via retinoic acid receptor-related orphan receptors α and γ

Journal: Toxicology

doi: 10.1016/j.tox.2015.01.007

Isoflavone-induced enhancement of RORα- and RORγ-mediated Il17a promoter activation in Jurkat cells. Cells were co-transfected with the pCMV-β-Gal and pCMV10-3xFlag-RORα (A) or pCMV10-3xFlag-RORγ (B) and pGL4.14 reporter plasmid under the control of the Il17a promoter and treated with increasing concentrations of the isoflavones at 0.1, 1, and 10 μM. After 24 h, relative LUC activity was determined as described in Materials and Methods. The firefly luciferase activity was normalized against β-galactosidase activity. BA, GE, FN, and DZ represent each isoflavone, biochanin A, genistein, formononetin, and daidzein, respectively. Values represent the means ± SEM ( n = 3). Significant differences from the vehicle control (DMSO) plus RORα or RORγ are indicated by asterisks (* P
Figure Legend Snippet: Isoflavone-induced enhancement of RORα- and RORγ-mediated Il17a promoter activation in Jurkat cells. Cells were co-transfected with the pCMV-β-Gal and pCMV10-3xFlag-RORα (A) or pCMV10-3xFlag-RORγ (B) and pGL4.14 reporter plasmid under the control of the Il17a promoter and treated with increasing concentrations of the isoflavones at 0.1, 1, and 10 μM. After 24 h, relative LUC activity was determined as described in Materials and Methods. The firefly luciferase activity was normalized against β-galactosidase activity. BA, GE, FN, and DZ represent each isoflavone, biochanin A, genistein, formononetin, and daidzein, respectively. Values represent the means ± SEM ( n = 3). Significant differences from the vehicle control (DMSO) plus RORα or RORγ are indicated by asterisks (* P

Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Activity Assay, Luciferase

Effects of isoflavones on interactions between ROR-LBD and the co-activator LXXLL peptide in mammalian two-hybrid assays. The analysis was performed by co-transfecting CHO-K1 cells with a pGL4.27-(UAS) 5 reporter plasmid, pCMV-β-Gal pM-EBIP96 peptide, and either VP16-RORα(LBD) (A) or VP16-RORγ(LBD) (B). Cells were treated in the presence of the vehicle (DMSO), or increasing concentrations of the four isoflavones as indicated. After 24 h, relative LUC activity was determined as described in Section 2. The firefly luciferase activity was normalized against β-galactosidase activity. BA, GE, FN, and DZ represent each isoflavone, biochanin A, genistein, formononetin, and daidzein, respectively. Values represent the means ± SEM ( n = 3) and are presented as the mean n -fold induction over the vehicle control. Significant differences from the vehicle control (DMSO) are indicated by asterisks (* P
Figure Legend Snippet: Effects of isoflavones on interactions between ROR-LBD and the co-activator LXXLL peptide in mammalian two-hybrid assays. The analysis was performed by co-transfecting CHO-K1 cells with a pGL4.27-(UAS) 5 reporter plasmid, pCMV-β-Gal pM-EBIP96 peptide, and either VP16-RORα(LBD) (A) or VP16-RORγ(LBD) (B). Cells were treated in the presence of the vehicle (DMSO), or increasing concentrations of the four isoflavones as indicated. After 24 h, relative LUC activity was determined as described in Section 2. The firefly luciferase activity was normalized against β-galactosidase activity. BA, GE, FN, and DZ represent each isoflavone, biochanin A, genistein, formononetin, and daidzein, respectively. Values represent the means ± SEM ( n = 3) and are presented as the mean n -fold induction over the vehicle control. Significant differences from the vehicle control (DMSO) are indicated by asterisks (* P

Techniques Used: Plasmid Preparation, Activity Assay, Luciferase

4) Product Images from "Cell-Specific Fine-Tuning of Neuronal Excitability by Differential Expression of Modulator Protein Isoforms"

Article Title: Cell-Specific Fine-Tuning of Neuronal Excitability by Differential Expression of Modulator Protein Isoforms

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1001-13.2013

The DPE and MTE are important regulators of transcription from the slob71 promoter. Core nucleotides within the MTE, DPE, or both MTE and DPE were mutated in the slob71 −1966 to +81 promoter. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. *** p
Figure Legend Snippet: The DPE and MTE are important regulators of transcription from the slob71 promoter. Core nucleotides within the MTE, DPE, or both MTE and DPE were mutated in the slob71 −1966 to +81 promoter. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. *** p

Techniques Used: Luciferase, Activity Assay, Transfection, Mutagenesis, Construct

Promoter elements in specific domains of slob71 affect transcriptional activity. A , Promoter fragments of slob71 were inserted upstream of a minP in the pGL4.23[ luc2 /minP] vector, which drives a low level of basal luciferase expression. Luciferase activity was measured in Drosophila S2 cells transfected with minP–luc or slob71 promoter fragment–minP–luc constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty minP–luc vector. B , C , Core nucleotides within the HB and MIRR recognition sites were mutated in slob71 promoters. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. Mutating MIRR or HB sites in the slob71 −1966 to +81 promoter increases relative luciferase activity ( B ). Mutation of the HB site in the slob71 −1966 to −1500 promoter fragment upstream of the minP increases relative luciferase activity, whereas mutation of the MIRR site has no effect ( C ). *** p
Figure Legend Snippet: Promoter elements in specific domains of slob71 affect transcriptional activity. A , Promoter fragments of slob71 were inserted upstream of a minP in the pGL4.23[ luc2 /minP] vector, which drives a low level of basal luciferase expression. Luciferase activity was measured in Drosophila S2 cells transfected with minP–luc or slob71 promoter fragment–minP–luc constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty minP–luc vector. B , C , Core nucleotides within the HB and MIRR recognition sites were mutated in slob71 promoters. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. Mutating MIRR or HB sites in the slob71 −1966 to +81 promoter increases relative luciferase activity ( B ). Mutation of the HB site in the slob71 −1966 to −1500 promoter fragment upstream of the minP increases relative luciferase activity, whereas mutation of the MIRR site has no effect ( C ). *** p

Techniques Used: Activity Assay, Plasmid Preparation, Luciferase, Expressing, Transfection, Construct, Mutagenesis

slob57 and slob71 promoters exhibit different transcriptional activity. Promoter regions upstream of the identified TSSs for slob57 and slob71 were cloned into the pGL4.10[ luc2 ] vector to drive the luciferase (luc) reporter gene. Drosophila S2 cells were transfected with various slob promoter–luc constructs and the pCMV–LacZ vector as an internal control. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty luc control vector. A , Summary of luciferase activity experiments with slob57 promoters. B , Summary of luciferase activity experiments with slob71 promoters. Relative luciferase activity driven by slob71 promoters is higher than that of slob57 . * p
Figure Legend Snippet: slob57 and slob71 promoters exhibit different transcriptional activity. Promoter regions upstream of the identified TSSs for slob57 and slob71 were cloned into the pGL4.10[ luc2 ] vector to drive the luciferase (luc) reporter gene. Drosophila S2 cells were transfected with various slob promoter–luc constructs and the pCMV–LacZ vector as an internal control. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty luc control vector. A , Summary of luciferase activity experiments with slob57 promoters. B , Summary of luciferase activity experiments with slob71 promoters. Relative luciferase activity driven by slob71 promoters is higher than that of slob57 . * p

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

5) Product Images from "The CCAAT/Enhancer-Binding Protein Beta-2 Isoform (CEBP?-2) Upregulates Galectin-7 Expression in Human Breast Cancer Cells"

Article Title: The CCAAT/Enhancer-Binding Protein Beta-2 Isoform (CEBP?-2) Upregulates Galectin-7 Expression in Human Breast Cancer Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0095087

C/EBPβ consensus binding site in galectin-7 promoter. ( A ) Schematic representation of C/EBP binding sites within the 5′ flanking region of the human galectin-7 gene. A s eries of 5′ deletion constructs of the 1500 bp galectin-7 promoter region was generated and cloned into the pGL3 Basic luciferase reporter vector. The resulting plasmids were transfected in MCF-7 and MDA-MB-468 cells. Locations of the putative C/EBP binding sites in the promoter, as determined using the TFsearch computational tool, are shown as empty boxes. ( B ) Sequence analysis of the C/EBP binding sites located at positions -105-98 and the -145-140. ( C ) Mutated constructs of the C/EBPβ binding sites on the 200 bp galectin-7 promoter region were generated and cloned into the pGL3 Basic luciferase reporter vector. The resulting plasmids were co-transfected in MCF-7 cells and were compared to the wild-type p200-galectin-7 promoter. Transfection efficiency was normalized by co-transfection with a β-galactosidase reporter vector.
Figure Legend Snippet: C/EBPβ consensus binding site in galectin-7 promoter. ( A ) Schematic representation of C/EBP binding sites within the 5′ flanking region of the human galectin-7 gene. A s eries of 5′ deletion constructs of the 1500 bp galectin-7 promoter region was generated and cloned into the pGL3 Basic luciferase reporter vector. The resulting plasmids were transfected in MCF-7 and MDA-MB-468 cells. Locations of the putative C/EBP binding sites in the promoter, as determined using the TFsearch computational tool, are shown as empty boxes. ( B ) Sequence analysis of the C/EBP binding sites located at positions -105-98 and the -145-140. ( C ) Mutated constructs of the C/EBPβ binding sites on the 200 bp galectin-7 promoter region were generated and cloned into the pGL3 Basic luciferase reporter vector. The resulting plasmids were co-transfected in MCF-7 cells and were compared to the wild-type p200-galectin-7 promoter. Transfection efficiency was normalized by co-transfection with a β-galactosidase reporter vector.

Techniques Used: Binding Assay, Construct, Generated, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Multiple Displacement Amplification, Sequencing, Cotransfection

6) Product Images from "Prospero-related homeobox 1 (Prox1) functions as a novel modulator of retinoic acid-related orphan receptors ?- and ?-mediated transactivation"

Article Title: Prospero-related homeobox 1 (Prox1) functions as a novel modulator of retinoic acid-related orphan receptors ?- and ?-mediated transactivation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt447

Both the N- and C-terminus of Prox1 are able to interact with RORγ. ( A ) MBP pull-down assays were performed using radiolabeled 35 S-RORγ (full-length), 35 S-RORγΔAF2 lacking the AF2 domain, 35 S-RORγΔLBD lacking the LBD and a series of MBP–Prox1 fragments, N(1-106), N(1-106)m, M(107-340), M(341-573), C(574-737) and C(574-737)m, as shown in the schematic. After incubation with amylose resin, MBP–Prox1 complexes were analyzed by PAGE, and radiolabeled RORγ was detected by autoradiography. Five percent of the input of each radiolabeled RORγ was loaded in the first lane. MBP was used as negative control. ( B ) MBP pull-down assays were performed using radiolabeled full-length RORγ ( 35 S-RORγ) and several N- and C-terminal fragments of Prox1, N(1-106), N(1-66), N(1-28), C(574-737), C(574-635) and C(636-737) as shown in the schematic. Samples were processed as described under A. ( C ) Loss of the N- and C-terminus of Prox1 diminishes its stabilizing effect on RORγ protein. HEK293 cells were transfected with pCMV10-3xFlag-RORγ and the pCMV-Myc-Prox1 or the pCMV-Myc-Prox1 mutant indicated and the level of RORγ and Myc-Prox1 protein examined by western blot analysis. Co-transfection with a β-Gal reporter indicated no significant difference in transfection efficiency between cells transfected with different Prox1 mutants.
Figure Legend Snippet: Both the N- and C-terminus of Prox1 are able to interact with RORγ. ( A ) MBP pull-down assays were performed using radiolabeled 35 S-RORγ (full-length), 35 S-RORγΔAF2 lacking the AF2 domain, 35 S-RORγΔLBD lacking the LBD and a series of MBP–Prox1 fragments, N(1-106), N(1-106)m, M(107-340), M(341-573), C(574-737) and C(574-737)m, as shown in the schematic. After incubation with amylose resin, MBP–Prox1 complexes were analyzed by PAGE, and radiolabeled RORγ was detected by autoradiography. Five percent of the input of each radiolabeled RORγ was loaded in the first lane. MBP was used as negative control. ( B ) MBP pull-down assays were performed using radiolabeled full-length RORγ ( 35 S-RORγ) and several N- and C-terminal fragments of Prox1, N(1-106), N(1-66), N(1-28), C(574-737), C(574-635) and C(636-737) as shown in the schematic. Samples were processed as described under A. ( C ) Loss of the N- and C-terminus of Prox1 diminishes its stabilizing effect on RORγ protein. HEK293 cells were transfected with pCMV10-3xFlag-RORγ and the pCMV-Myc-Prox1 or the pCMV-Myc-Prox1 mutant indicated and the level of RORγ and Myc-Prox1 protein examined by western blot analysis. Co-transfection with a β-Gal reporter indicated no significant difference in transfection efficiency between cells transfected with different Prox1 mutants.

Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography, Negative Control, Transfection, Mutagenesis, Western Blot, Cotransfection

Prox1 represses RORγ- and RORα-mediated transcriptional activation. ( A ) Huh-7 cells were co-transfected with the pGL4.27-(RORE) 5 or pGL4.10- Npas2 (-1534/+81) reporter plasmid, pCMV-β-Gal, pCMV10-3xFlag-RORγ or -RORα, and increasing amounts of pCMV-Myc-Prox1 expression plasmid (ROR:Prox1 = 1:0.2, 1:0.5, 1:1). Luciferase and β-galactosidase activities were measured 24 h later. ( B ) Huh-7 cells were co-transfected with pGL4.10 reporter plasmid containing Npas2 promoter region (−1534/+81) and expression vectors as described earlier in the text. ( C ) Mammalian monohybrid analysis. CHO cells were transfected with pGL4.27-(UAS) 5 reporter plasmid containing UAS, pCMV-β-Gal, pM-RORγLBD and increasing amounts of pCMV-Myc-Prox1 expression plasmid (ROR:Prox1 ratios are 1:0.1, 1:0.2, 1:0.5 and 1:1). ( D ) Mammalian two-hybrid analysis. CHO cells were transfected with pGL4.27-(UAS) 5 , pCMV-β-Gal, pM-GAL4-LXXLL(EBIP96), VP16-RORγ(LBD) or -RORα(LBD) and increasing amounts of pCMV-Myc-Prox1 (ROR:Prox1 = 1:0.2, 1:0.5, 1:1). Luciferase and β-galactosidase activities were measured 24 h later. ( E ) Schematic presentation of several N- or C-terminal Prox1 deletion constructs and mutants containing mutations in the LXXLL motifs or the homeodomain. ( F ) Huh-7 cells were co-transfected with pGL4.10- Npas2 (-1534/+81), pCMV-β-Gal, pCMV10-3xFlag-RORγ and pCMV-Myc expression vector containing Prox1 or the Prox1 mutants P(LXXLL)m, PΔN106, P(HD)m, PΔN106(HD)m, PΔC573, PΔC636 and PΔN106ΔC636) as indicated. ( G ) Mammalian two-hybrid analysis. CHO cells were transfected with pGL4.27-(UAS) 5 , pCMV-β-Gal, pM-GAL4-LXXLL(EBIP96), VP16-RORγ(LBD) and pCMV-Myc expression vector containing Prox1, PΔN106 or PΔC636. ( H ) The region of Prox1 between amino acids 723 and 729 is required for its repression of RORγ-mediated transactivation of Npas2 (−1534/+81). Huh-7 cells were transfected with pGL4.10- Npas2 (-1534/+81), pCMV-β-Gal, pCMV10-3xFlag-RORγ and pCMV-Myc expression vector containing Prox1, PΔN106, PΔN106ΔC729, PΔN106ΔC722, PΔN106ΔC716, PΔN106ΔC686 or PΔN106ΔC636, as indicated. Luciferase and β-galactosidase activities were measured 24 h later. All the experiments were performed in triplicate. Data represent mean ± SEM; * P
Figure Legend Snippet: Prox1 represses RORγ- and RORα-mediated transcriptional activation. ( A ) Huh-7 cells were co-transfected with the pGL4.27-(RORE) 5 or pGL4.10- Npas2 (-1534/+81) reporter plasmid, pCMV-β-Gal, pCMV10-3xFlag-RORγ or -RORα, and increasing amounts of pCMV-Myc-Prox1 expression plasmid (ROR:Prox1 = 1:0.2, 1:0.5, 1:1). Luciferase and β-galactosidase activities were measured 24 h later. ( B ) Huh-7 cells were co-transfected with pGL4.10 reporter plasmid containing Npas2 promoter region (−1534/+81) and expression vectors as described earlier in the text. ( C ) Mammalian monohybrid analysis. CHO cells were transfected with pGL4.27-(UAS) 5 reporter plasmid containing UAS, pCMV-β-Gal, pM-RORγLBD and increasing amounts of pCMV-Myc-Prox1 expression plasmid (ROR:Prox1 ratios are 1:0.1, 1:0.2, 1:0.5 and 1:1). ( D ) Mammalian two-hybrid analysis. CHO cells were transfected with pGL4.27-(UAS) 5 , pCMV-β-Gal, pM-GAL4-LXXLL(EBIP96), VP16-RORγ(LBD) or -RORα(LBD) and increasing amounts of pCMV-Myc-Prox1 (ROR:Prox1 = 1:0.2, 1:0.5, 1:1). Luciferase and β-galactosidase activities were measured 24 h later. ( E ) Schematic presentation of several N- or C-terminal Prox1 deletion constructs and mutants containing mutations in the LXXLL motifs or the homeodomain. ( F ) Huh-7 cells were co-transfected with pGL4.10- Npas2 (-1534/+81), pCMV-β-Gal, pCMV10-3xFlag-RORγ and pCMV-Myc expression vector containing Prox1 or the Prox1 mutants P(LXXLL)m, PΔN106, P(HD)m, PΔN106(HD)m, PΔC573, PΔC636 and PΔN106ΔC636) as indicated. ( G ) Mammalian two-hybrid analysis. CHO cells were transfected with pGL4.27-(UAS) 5 , pCMV-β-Gal, pM-GAL4-LXXLL(EBIP96), VP16-RORγ(LBD) and pCMV-Myc expression vector containing Prox1, PΔN106 or PΔC636. ( H ) The region of Prox1 between amino acids 723 and 729 is required for its repression of RORγ-mediated transactivation of Npas2 (−1534/+81). Huh-7 cells were transfected with pGL4.10- Npas2 (-1534/+81), pCMV-β-Gal, pCMV10-3xFlag-RORγ and pCMV-Myc expression vector containing Prox1, PΔN106, PΔN106ΔC729, PΔN106ΔC722, PΔN106ΔC716, PΔN106ΔC686 or PΔN106ΔC636, as indicated. Luciferase and β-galactosidase activities were measured 24 h later. All the experiments were performed in triplicate. Data represent mean ± SEM; * P

Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Construct

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Transfection:

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Luciferase:

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Article Title: Transforming growth factor ?-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells
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Article Title: Expression of Galectin-7 Is Induced in Breast Cancer Cells by Mutant p53
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Enzymatic Assay:

Article Title: Expression of Galectin-7 Is Induced in Breast Cancer Cells by Mutant p53
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Cell Culture:

Article Title: Combinatorial regulation of endothelial gene expression by ets and forkhead transcription factors
Article Snippet: .. Following transfection, cells were cultured for 48 h, then harvested and assayed using the Luminescent β-galactosidase Detection kit II (Clontech), as previously described ( ). .. For ChIP, primary mouse embryo fibroblasts (MEFs) were transfected using Lipofectamine LTX and 8 μg of either pcDNA3.1-FLAG-FoxC2 or empty pCDNA3.1 vector in 10 cm dishes.

Incubation:

Article Title: Transforming growth factor ?-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells
Article Snippet: .. After 20 hr incubation in the absence or presence of 240 pM TGF-β1, luciferase activity was determined using the luciferase assay system (Promega) and β-galactosidase activity was measured using the Luminescent β-galactosidase detection kit (CLONTECH) with an Analytical Luminescent Laboratory luminometer. .. To address the physiological relevance of the Smad3 protein in cellular responses to TGF-β, we introduced the wild-type and various mutant Smad3 genes into a novel retroviral vector.

Activity Assay:

Article Title: Polymorphism of the human ?1 immunoglobulin gene 3? enhancer hs1,2 and its relation to gene expression
Article Snippet: .. After 20 hr of culture in RPMI-1640 medium (Gibco, Cergy Pontoise, France) supplemented with 10% fetal calf serum (FCS), 2 m m l -glutamine, 100 µg/ml penicillin and 10 µg/ml streptomycin, cells were recovered, lysed and assayed for luciferase activity using the Luclite Plus Assay Kit (Packard Bio-Science B.V., Groningen, The Netherlands) and for β galactosidase activity with the luminescent β galactosidase detection kit (Clontech, Palo Alto, CA) to standardize the assay. ..

Article Title: Transforming growth factor ?-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells
Article Snippet: .. After 20 hr incubation in the absence or presence of 240 pM TGF-β1, luciferase activity was determined using the luciferase assay system (Promega) and β-galactosidase activity was measured using the Luminescent β-galactosidase detection kit (CLONTECH) with an Analytical Luminescent Laboratory luminometer. .. To address the physiological relevance of the Smad3 protein in cellular responses to TGF-β, we introduced the wild-type and various mutant Smad3 genes into a novel retroviral vector.

Article Title: Cooperative activation of cardiac transcription through myocardin bridging of paired MEF2 sites
Article Snippet: .. Cells were harvested 48 h post-transfection, and cellular extracts were prepared and assayed for β-galactosidase activity using the Luminescent β-galactosidase Detection Kit (Clontech) as previously described ( ). ..

Article Title: Mesenchymal stem cells decrease lung inflammation during sepsis, acting through inhibition of the MAPK pathway
Article Snippet: .. Luciferase values were normalized using β-galactosidase, and activity measured with the Luminescent β-Galactosidase Detection Kit II (CLONTECH, Palo Alto, CA, USA). ..

Article Title: Expression of Galectin-7 Is Induced in Breast Cancer Cells by Mutant p53
Article Snippet: .. Luciferase activity was measured using the Luciferase Assay System protocol (Promega, Madison, WI, USA) and a luminometer (Lumat LB 9507, Berthold). β-galactosidase activity was measured using a colorimetric enzyme assay using the Luminescent β-Galactosidase Detection Kit II according to the manufacturer’s instructions (Clontech Laboratories, Mountain View, CA). .. Luciferase expression levels were normalized to the levels of β-galactosidase expression.

Chromatin Immunoprecipitation:

Article Title: HIF2α signaling inhibits adherens junctional disruption in acute lung injury
Article Snippet: .. Anti–VE-cadherin (sc-9989, sc-6458, and sc-52751), anti–VE-PTP (SC-28905), anti-HIF2α (sc-13596), anti-PHD2 (sc-271835), anti-sFLT (sc-9029), and anti–β-actin (sc-1616) antibodies were purchased from Santa Cruz Biotechnology Inc.; mouse monoclonal anti–VE-PTP (610180) and anti-HIF1α (610959) antibodies were from BD; anti-HIF3α (ab10134) antibody was purchased from Abcam; anti-HIF2α (NB100-122) antibody was from Novus Biologicals; Lipofectamine 2000, ViraPower Lentiviral Expression System, and Alexa Fluor 488–, 594–, and 633–conjugated secondary antibodies and Alexa Fluor 555–albumin were obtained from Invitrogen; anti–VE-cadherin pY658, pY695, and pY731 antibodies, anti-phosphotyrosine antibody (4G10), and the ChIP assay kit were from EMD Millipore; the luciferase assay kit was purchased from Promega; and the Luminescent β-galactosidase Detection Kit was from Clontech. .. For in vivo experiments, we used EC-specific inducible Hif2a–/– mice generated by i . p . administration of tamoxifen (2 mg/day for 5 days) to Tie2-Cre Hif2afl/fl mice (129/B6 background), in which tamoxifen induced expression of a fusion protein of Cre recombinase with the modified estrogen receptor–binding domain ( CreERT2 ) under the control of the Tie2 promoter ( , ).

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    TaKaRa vector expressing β galactosidase
    The DPE and MTE are important regulators of transcription from the slob71 promoter. Core nucleotides within the MTE, DPE, or both MTE and DPE were mutated in the slob71 −1966 to +81 promoter. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to <t>β-gal</t> activity and is reported as the percentage of activity exhibited by the intact control construct. *** p
    Vector Expressing β Galactosidase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vector expressing β galactosidase/product/TaKaRa
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    vector expressing β galactosidase - by Bioz Stars, 2020-09
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    91
    TaKaRa beta galactosidase β gal reporter plasmid pcmv lacz
    The DPE and MTE are important regulators of transcription from the slob71 promoter. Core nucleotides within the MTE, DPE, or both MTE and DPE were mutated in the slob71 −1966 to +81 promoter. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to <t>β-gal</t> activity and is reported as the percentage of activity exhibited by the intact control construct. *** p
    Beta Galactosidase β Gal Reporter Plasmid Pcmv Lacz, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/beta galactosidase β gal reporter plasmid pcmv lacz/product/TaKaRa
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    beta galactosidase β gal reporter plasmid pcmv lacz - by Bioz Stars, 2020-09
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    The DPE and MTE are important regulators of transcription from the slob71 promoter. Core nucleotides within the MTE, DPE, or both MTE and DPE were mutated in the slob71 −1966 to +81 promoter. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. *** p

    Journal: The Journal of Neuroscience

    Article Title: Cell-Specific Fine-Tuning of Neuronal Excitability by Differential Expression of Modulator Protein Isoforms

    doi: 10.1523/JNEUROSCI.1001-13.2013

    Figure Lengend Snippet: The DPE and MTE are important regulators of transcription from the slob71 promoter. Core nucleotides within the MTE, DPE, or both MTE and DPE were mutated in the slob71 −1966 to +81 promoter. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. *** p

    Article Snippet: S2 cells were transfected with the luciferase reporter constructs (2 μg/well) along with a vector expressing β-galactosidase (β-gal; pCMV–LacZ vector from Clontech; 1 μg/well) in duplicate using Lipofectamine (Invitrogen).

    Techniques: Luciferase, Activity Assay, Transfection, Mutagenesis, Construct

    Promoter elements in specific domains of slob71 affect transcriptional activity. A , Promoter fragments of slob71 were inserted upstream of a minP in the pGL4.23[ luc2 /minP] vector, which drives a low level of basal luciferase expression. Luciferase activity was measured in Drosophila S2 cells transfected with minP–luc or slob71 promoter fragment–minP–luc constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty minP–luc vector. B , C , Core nucleotides within the HB and MIRR recognition sites were mutated in slob71 promoters. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. Mutating MIRR or HB sites in the slob71 −1966 to +81 promoter increases relative luciferase activity ( B ). Mutation of the HB site in the slob71 −1966 to −1500 promoter fragment upstream of the minP increases relative luciferase activity, whereas mutation of the MIRR site has no effect ( C ). *** p

    Journal: The Journal of Neuroscience

    Article Title: Cell-Specific Fine-Tuning of Neuronal Excitability by Differential Expression of Modulator Protein Isoforms

    doi: 10.1523/JNEUROSCI.1001-13.2013

    Figure Lengend Snippet: Promoter elements in specific domains of slob71 affect transcriptional activity. A , Promoter fragments of slob71 were inserted upstream of a minP in the pGL4.23[ luc2 /minP] vector, which drives a low level of basal luciferase expression. Luciferase activity was measured in Drosophila S2 cells transfected with minP–luc or slob71 promoter fragment–minP–luc constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty minP–luc vector. B , C , Core nucleotides within the HB and MIRR recognition sites were mutated in slob71 promoters. Luciferase activity was measured in Drosophila S2 cells transfected with mutant or intact control constructs. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the percentage of activity exhibited by the intact control construct. Mutating MIRR or HB sites in the slob71 −1966 to +81 promoter increases relative luciferase activity ( B ). Mutation of the HB site in the slob71 −1966 to −1500 promoter fragment upstream of the minP increases relative luciferase activity, whereas mutation of the MIRR site has no effect ( C ). *** p

    Article Snippet: S2 cells were transfected with the luciferase reporter constructs (2 μg/well) along with a vector expressing β-galactosidase (β-gal; pCMV–LacZ vector from Clontech; 1 μg/well) in duplicate using Lipofectamine (Invitrogen).

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

    slob57 and slob71 promoters exhibit different transcriptional activity. Promoter regions upstream of the identified TSSs for slob57 and slob71 were cloned into the pGL4.10[ luc2 ] vector to drive the luciferase (luc) reporter gene. Drosophila S2 cells were transfected with various slob promoter–luc constructs and the pCMV–LacZ vector as an internal control. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty luc control vector. A , Summary of luciferase activity experiments with slob57 promoters. B , Summary of luciferase activity experiments with slob71 promoters. Relative luciferase activity driven by slob71 promoters is higher than that of slob57 . * p

    Journal: The Journal of Neuroscience

    Article Title: Cell-Specific Fine-Tuning of Neuronal Excitability by Differential Expression of Modulator Protein Isoforms

    doi: 10.1523/JNEUROSCI.1001-13.2013

    Figure Lengend Snippet: slob57 and slob71 promoters exhibit different transcriptional activity. Promoter regions upstream of the identified TSSs for slob57 and slob71 were cloned into the pGL4.10[ luc2 ] vector to drive the luciferase (luc) reporter gene. Drosophila S2 cells were transfected with various slob promoter–luc constructs and the pCMV–LacZ vector as an internal control. The relative luciferase activity is the luciferase activity normalized to β-gal activity and is reported as the fold change compared with the empty luc control vector. A , Summary of luciferase activity experiments with slob57 promoters. B , Summary of luciferase activity experiments with slob71 promoters. Relative luciferase activity driven by slob71 promoters is higher than that of slob57 . * p

    Article Snippet: S2 cells were transfected with the luciferase reporter constructs (2 μg/well) along with a vector expressing β-galactosidase (β-gal; pCMV–LacZ vector from Clontech; 1 μg/well) in duplicate using Lipofectamine (Invitrogen).

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