Structured Review

Promega β galactosidase expression plasmid
Influence of MBD2 and TACC3 over-expression on activity of methylated transfected or endogenous galectin-1 promoter activity. ( A ) 293T cells were transfected with 3 µg of unmethylated (gray bar) or in vitro methylated (black bars) pGAT1700 reporter plasmid. Cotransfected expression plasmids were pSVβ (2 µg) and, where indicated, pMyc-MBD2 (2 µg) and pHA-TACC3 at different amounts. The level of transcription is given as a percentage of CAT activity seen in the presence of unmethylated pGAT1700 reporter plasmid alone (set as 100%). Percent relative activity of the promoters is expressed as the ratio of CAT to <t>β-galactosidase</t> activity. The results are presented as an average of multiple experiments +/− SD. Each histogram bar represents the mean of at least four independent transfections. ( B ). Quantitative RT–PCR assays of endogenous galectin-1 mRNAs were performed on PC Cl3 cells untreated or treated with the indicated concentrations of 5-aza-2′deoxycytidine (Aza) or Trycostatin A (TSA) for 36 h. Where indicated (TACC3), cells were transfected with pHA-TACC3 expression vector. mRNA expression levels are shown as fold-induction compared with the PC Cl3 value, arbitrarily set as 1. The results are presented as an average of at least three independent experiments +/− SD.
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Images

1) Product Images from "TACC3 mediates the association of MBD2 with histone acetyltransferases and relieves transcriptional repression of methylated promoters"

Article Title: TACC3 mediates the association of MBD2 with histone acetyltransferases and relieves transcriptional repression of methylated promoters

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkj400

Influence of MBD2 and TACC3 over-expression on activity of methylated transfected or endogenous galectin-1 promoter activity. ( A ) 293T cells were transfected with 3 µg of unmethylated (gray bar) or in vitro methylated (black bars) pGAT1700 reporter plasmid. Cotransfected expression plasmids were pSVβ (2 µg) and, where indicated, pMyc-MBD2 (2 µg) and pHA-TACC3 at different amounts. The level of transcription is given as a percentage of CAT activity seen in the presence of unmethylated pGAT1700 reporter plasmid alone (set as 100%). Percent relative activity of the promoters is expressed as the ratio of CAT to β-galactosidase activity. The results are presented as an average of multiple experiments +/− SD. Each histogram bar represents the mean of at least four independent transfections. ( B ). Quantitative RT–PCR assays of endogenous galectin-1 mRNAs were performed on PC Cl3 cells untreated or treated with the indicated concentrations of 5-aza-2′deoxycytidine (Aza) or Trycostatin A (TSA) for 36 h. Where indicated (TACC3), cells were transfected with pHA-TACC3 expression vector. mRNA expression levels are shown as fold-induction compared with the PC Cl3 value, arbitrarily set as 1. The results are presented as an average of at least three independent experiments +/− SD.
Figure Legend Snippet: Influence of MBD2 and TACC3 over-expression on activity of methylated transfected or endogenous galectin-1 promoter activity. ( A ) 293T cells were transfected with 3 µg of unmethylated (gray bar) or in vitro methylated (black bars) pGAT1700 reporter plasmid. Cotransfected expression plasmids were pSVβ (2 µg) and, where indicated, pMyc-MBD2 (2 µg) and pHA-TACC3 at different amounts. The level of transcription is given as a percentage of CAT activity seen in the presence of unmethylated pGAT1700 reporter plasmid alone (set as 100%). Percent relative activity of the promoters is expressed as the ratio of CAT to β-galactosidase activity. The results are presented as an average of multiple experiments +/− SD. Each histogram bar represents the mean of at least four independent transfections. ( B ). Quantitative RT–PCR assays of endogenous galectin-1 mRNAs were performed on PC Cl3 cells untreated or treated with the indicated concentrations of 5-aza-2′deoxycytidine (Aza) or Trycostatin A (TSA) for 36 h. Where indicated (TACC3), cells were transfected with pHA-TACC3 expression vector. mRNA expression levels are shown as fold-induction compared with the PC Cl3 value, arbitrarily set as 1. The results are presented as an average of at least three independent experiments +/− SD.

Techniques Used: Over Expression, Activity Assay, Methylation, Transfection, In Vitro, Plasmid Preparation, Expressing, Quantitative RT-PCR

Definition of MBD2 and TACC3 binding region in yeast. ( A ) Schematic representation of structural and functional domains of MBD2 and TACC3 proteins. For MBD2, the methyl-binding-domain (MBD) is indicated as a black box; the putative coiled–coil domain is indicated as a gray box. For TACC3, the striped box contains a conserved N-terminal region and the TACC domain is indicated as a gray box. TACC3-mir (minimal interacting region) represents the minimal TACC3 region interacting with MBD2 in yeast. ( B ) Mapping of the TACC3-binding region of MBD2 by yeast two-hybrid experiments. Full length and deletion mutants of MBD2 were fused to GAL4 DNA binding domain (bait) and the TACC3-mir (amino acids from 571 to 837) was fused to the GAL4 activation domain (pGAD-F3 plasmid clone). The interaction between the bait proteins and prey was tested on the basis of the His and Ade reporter gene activation and by quantitative liquid β-galactosidase assay.
Figure Legend Snippet: Definition of MBD2 and TACC3 binding region in yeast. ( A ) Schematic representation of structural and functional domains of MBD2 and TACC3 proteins. For MBD2, the methyl-binding-domain (MBD) is indicated as a black box; the putative coiled–coil domain is indicated as a gray box. For TACC3, the striped box contains a conserved N-terminal region and the TACC domain is indicated as a gray box. TACC3-mir (minimal interacting region) represents the minimal TACC3 region interacting with MBD2 in yeast. ( B ) Mapping of the TACC3-binding region of MBD2 by yeast two-hybrid experiments. Full length and deletion mutants of MBD2 were fused to GAL4 DNA binding domain (bait) and the TACC3-mir (amino acids from 571 to 837) was fused to the GAL4 activation domain (pGAD-F3 plasmid clone). The interaction between the bait proteins and prey was tested on the basis of the His and Ade reporter gene activation and by quantitative liquid β-galactosidase assay.

Techniques Used: Binding Assay, Functional Assay, Activation Assay, Plasmid Preparation

2) Product Images from "Glucocorticoids Regulate the Expression of the Human Osteoblastic Endothelin A Receptor Gene "

Article Title: Glucocorticoids Regulate the Expression of the Human Osteoblastic Endothelin A Receptor Gene

Journal: The Journal of Experimental Medicine

doi:

( A ) Schematic demonstration of the chimeric construct of the 5′-flanking region of the ETR A gene fused to luciferase gene in pGL3 Basic vector. ( B ) Reporter gene activity was assayed in HOC cotransfected with reporter gene construct and glucocorticoid receptor expression plasmid and β-galactosidase expression plasmid, treated for 24 h in parallel with 100 nM dexamethasone and control medium, and expressed in corrected luciferase activity ± SE.
Figure Legend Snippet: ( A ) Schematic demonstration of the chimeric construct of the 5′-flanking region of the ETR A gene fused to luciferase gene in pGL3 Basic vector. ( B ) Reporter gene activity was assayed in HOC cotransfected with reporter gene construct and glucocorticoid receptor expression plasmid and β-galactosidase expression plasmid, treated for 24 h in parallel with 100 nM dexamethasone and control medium, and expressed in corrected luciferase activity ± SE.

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

3) Product Images from "Evidence against a Role for ?-Arrestin1 in STAT1 Dephosphorylation and the Inhibition of Interferon-? Signaling"

Article Title: Evidence against a Role for ?-Arrestin1 in STAT1 Dephosphorylation and the Inhibition of Interferon-? Signaling

Journal: Molecular Cell

doi: 10.1016/j.molcel.2013.02.024

Cotransfection of β-Arrestin1 Has Nonspecific Inhibitory Effects on Gene Transcription (A and B) Interferon-γ-induced luciferase reporter activity in untreated and IFNγ-treated HeLa cells using increasing amounts of two β-arrestin1 constructs as indicated. Error bars represent means ± standard deviation for one representative experiment done in triplicates. The respective values for “fold induction” (induced/uninduced) are shown in the panels on the right. (C) Immunoblot showing the effects of FLAG-tagged β-arrestin1 on coexpressed β-galactosidase in HeLa cells. After western blotting, the nitrocellulose membrane was cut horizontally at the ∼80 kDa position. The upper membrane was probed with antibody against β-galactosidase; the other was consecutively probed with anti-FLAG-tag and anti-β-actin. (D and E) Interferon-γ-induced luciferase reporter activity in untreated and IFNγ-treated HeLa cells. The cells were transfected with plasmid pBS (control) or the expression plasmid for GFP-tagged β-arrestin1, and in addition with different ratios of plasmids encoding β-galactosidase and the luciferase reporter gene as indicated. Error bars represent means ± standard deviation of one experiment done in triplicate. The respective values for “fold induction” (induced/uninduced) are shown in (E).
Figure Legend Snippet: Cotransfection of β-Arrestin1 Has Nonspecific Inhibitory Effects on Gene Transcription (A and B) Interferon-γ-induced luciferase reporter activity in untreated and IFNγ-treated HeLa cells using increasing amounts of two β-arrestin1 constructs as indicated. Error bars represent means ± standard deviation for one representative experiment done in triplicates. The respective values for “fold induction” (induced/uninduced) are shown in the panels on the right. (C) Immunoblot showing the effects of FLAG-tagged β-arrestin1 on coexpressed β-galactosidase in HeLa cells. After western blotting, the nitrocellulose membrane was cut horizontally at the ∼80 kDa position. The upper membrane was probed with antibody against β-galactosidase; the other was consecutively probed with anti-FLAG-tag and anti-β-actin. (D and E) Interferon-γ-induced luciferase reporter activity in untreated and IFNγ-treated HeLa cells. The cells were transfected with plasmid pBS (control) or the expression plasmid for GFP-tagged β-arrestin1, and in addition with different ratios of plasmids encoding β-galactosidase and the luciferase reporter gene as indicated. Error bars represent means ± standard deviation of one experiment done in triplicate. The respective values for “fold induction” (induced/uninduced) are shown in (E).

Techniques Used: Cotransfection, Luciferase, Activity Assay, Construct, Standard Deviation, Western Blot, FLAG-tag, Transfection, Plasmid Preparation, Expressing

β-Arrestin1 Does Not Affect STAT1 Phosphorylation (A) Immunoblot showing a time course of STAT1 phosphorylation in IFNγ-treated HeLa cells that transiently express β-arrestin1 or β-galactosidase as indicated. Cells were left untreated or treated with IFNγ for 60 min, followed by incubation for 0–240 min in growth medium (GM) without interferon. Probing was done first with anti-Tyr701-phoshorylated STAT1 antibody, then with a mixture of anti-human STAT1 and anti-β-actin antibody, followed by a mixture of anti-GFP and anti-FLAG-tag, and finally with anti-β-galactosidase. Molecular weight marker positions are indicated. The diagram combines the results of this and another experiment. It shows specific Tyr701-phosphorylation of STAT1 under the different experimental conditions (specific Tyr701 phosphorylation after 60 min IFNγ was set as 100). Results were calculated as the mean ± standard deviation. (B) Shown are immunoblot analyses of wild-type (WT) and β-arrestin1-deficient MEFs using anti-β-arrestin1 antibody. Reprobing was done with anti-β-actin. Positions of molecular weight markers and β-arrestin 1 are indicated. (C) Top, immunoblot analyses of STAT1 dephosphorylation in WT and mutant MEFs. Bottom, diagram combining the results of this and two additional experiments giving specific Tyr701 phosphorylation of STAT1. The value for 60 min IFNγ was set as 100. Results were calculated as the mean ± standard deviation.
Figure Legend Snippet: β-Arrestin1 Does Not Affect STAT1 Phosphorylation (A) Immunoblot showing a time course of STAT1 phosphorylation in IFNγ-treated HeLa cells that transiently express β-arrestin1 or β-galactosidase as indicated. Cells were left untreated or treated with IFNγ for 60 min, followed by incubation for 0–240 min in growth medium (GM) without interferon. Probing was done first with anti-Tyr701-phoshorylated STAT1 antibody, then with a mixture of anti-human STAT1 and anti-β-actin antibody, followed by a mixture of anti-GFP and anti-FLAG-tag, and finally with anti-β-galactosidase. Molecular weight marker positions are indicated. The diagram combines the results of this and another experiment. It shows specific Tyr701-phosphorylation of STAT1 under the different experimental conditions (specific Tyr701 phosphorylation after 60 min IFNγ was set as 100). Results were calculated as the mean ± standard deviation. (B) Shown are immunoblot analyses of wild-type (WT) and β-arrestin1-deficient MEFs using anti-β-arrestin1 antibody. Reprobing was done with anti-β-actin. Positions of molecular weight markers and β-arrestin 1 are indicated. (C) Top, immunoblot analyses of STAT1 dephosphorylation in WT and mutant MEFs. Bottom, diagram combining the results of this and two additional experiments giving specific Tyr701 phosphorylation of STAT1. The value for 60 min IFNγ was set as 100. Results were calculated as the mean ± standard deviation.

Techniques Used: Incubation, FLAG-tag, Molecular Weight, Marker, Standard Deviation, De-Phosphorylation Assay, Mutagenesis

4) Product Images from "A Novel Function of Apolipoprotein E: Upregulation of ATP-Binding Cassette Transporter A1 Expression"

Article Title: A Novel Function of Apolipoprotein E: Upregulation of ATP-Binding Cassette Transporter A1 Expression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0021453

The effect of ApoE on lipoprotein-induced ABCA1 promoter activity. Pane A: Two constructs containing wild-type and Sp1-binding site-mutated (Mut-Sp1) ABCA1 promoter region were subcloned into a luciferase (Luc) reporter plasmid (pGL-2). The mutated sequences in the Mut-Sp1 construct are shown in the schematic diagram. Panel B: Mouse macrophages were transfected with a β-galactosidase expression plasmid and the ABCA1 promoter-reporter constructs or the empty pGL-2 vector. The transfected macrophages were treated with 20 µg/ml of wild-type (E + /B), ApoE-free (EÎ/B) or ApoE3-enriched (E3/B) lipoproteins, or culture medium alone (control) for 4 hrs. Luciferase activity was normalized to the β-galactosidase activity and expressed relative to that of pGL2 basic vector. Values represent the mean ± SEM of five independent experiments. * P
Figure Legend Snippet: The effect of ApoE on lipoprotein-induced ABCA1 promoter activity. Pane A: Two constructs containing wild-type and Sp1-binding site-mutated (Mut-Sp1) ABCA1 promoter region were subcloned into a luciferase (Luc) reporter plasmid (pGL-2). The mutated sequences in the Mut-Sp1 construct are shown in the schematic diagram. Panel B: Mouse macrophages were transfected with a β-galactosidase expression plasmid and the ABCA1 promoter-reporter constructs or the empty pGL-2 vector. The transfected macrophages were treated with 20 µg/ml of wild-type (E + /B), ApoE-free (EÎ/B) or ApoE3-enriched (E3/B) lipoproteins, or culture medium alone (control) for 4 hrs. Luciferase activity was normalized to the β-galactosidase activity and expressed relative to that of pGL2 basic vector. Values represent the mean ± SEM of five independent experiments. * P

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

5) Product Images from "Stimulation of NF-?B Activity by the HIV Restriction Factor BST2"

Article Title: Stimulation of NF-?B Activity by the HIV Restriction Factor BST2

Journal: Journal of Virology

doi: 10.1128/JVI.02272-12

BST2 induces NF-κB activity. (A) BST2-mediated induction of NF-κB activity compared to the induction of AP-1 activity and expression driven by an interferon-stimulated response element (ISRE). HEK293T cells were transfected in wells of a 24-well plate with plasmids encoding firefly luciferase under the control of promoters responsive to the various transcription factors (200 ng) together with increasing amounts of a plasmid expressing BST2; a plasmid expressing β-galactosidase (20 ng) was included as a normalization control. The next day, the cells were lysed and luciferase and β-galactosidase activities were measured by luminometry. Data are the ratio of relative light units (RLU) measured for luciferase activity to RLU measured for β-galactosidase activity. (B) Controls for the various indicators shown as fold inductions compared to the indicator alone. A plasmid expressing TGF-β-activated kinase 1 (TAK1) was used at a 30-ng or 680-ng amount, as indicated. TNF-α (TNF) was used at 1 ng/ml, and phorbol myristic acetate (PMA) was used at 100 ng/ml. A plasmid expressing IRF-3 5D was used (400 ng) as a positive control for the ISRE indicator. Data are the average of duplicates.
Figure Legend Snippet: BST2 induces NF-κB activity. (A) BST2-mediated induction of NF-κB activity compared to the induction of AP-1 activity and expression driven by an interferon-stimulated response element (ISRE). HEK293T cells were transfected in wells of a 24-well plate with plasmids encoding firefly luciferase under the control of promoters responsive to the various transcription factors (200 ng) together with increasing amounts of a plasmid expressing BST2; a plasmid expressing β-galactosidase (20 ng) was included as a normalization control. The next day, the cells were lysed and luciferase and β-galactosidase activities were measured by luminometry. Data are the ratio of relative light units (RLU) measured for luciferase activity to RLU measured for β-galactosidase activity. (B) Controls for the various indicators shown as fold inductions compared to the indicator alone. A plasmid expressing TGF-β-activated kinase 1 (TAK1) was used at a 30-ng or 680-ng amount, as indicated. TNF-α (TNF) was used at 1 ng/ml, and phorbol myristic acetate (PMA) was used at 100 ng/ml. A plasmid expressing IRF-3 5D was used (400 ng) as a positive control for the ISRE indicator. Data are the average of duplicates.

Techniques Used: Activity Assay, Expressing, Transfection, Luciferase, Plasmid Preparation, Positive Control

The YxY sequence in the BST2 cytoplasmic domain is required for signaling but dispensable for restriction. (A) HEK293T cells were transfected in wells of a 24-well plate with a plasmid encoding firefly luciferase under the control of a minimal promoter containing binding sites for NF-κB (200 ng) along with increasing amounts of a plasmid expressing BST2 (indicated); a plasmid expressing β-galactosidase (220 ng) was included as a normalization control. The next day, the cells were lysed and luciferase and β-galactosidase activities were measured by luminometry. Data are the ratio of relative light units (RLU) measured for luciferase activity to RLU measured for β-galactosidase activity and are the average of duplicates. (B) Restriction of HIV-1 virion release by BST2. HEK293T cells were transfected in wells of a 12-well plate with a plasmid encoding an HIV-1 provirus lacking vpu (1.6 μg) along with a plasmid encoding either no BST2, wild-type BST2, or the indicated mutants (30 ng). The next day, the supernatants were removed and the virions pelleted though a 20% sucrose cushion and then resuspended in the original culture volume before measurement of p24 Gag (capsid) concentration by ELISA. Data are the average of duplicates. (C) Cells from the experiment shown in panel B were analyzed by SDS-PAGE and immunoblotting for BST2 and p55 Gag precursor. In all panels, Y6/8A is a BST2 mutant in which the tyrosines at positions 6 and 8 are replaced with alanines, STS is a mutant in which the serine and threonine residues at positions 3 to 5 are replaced with alanines, and KKCC is a mutant in which the lysine residues at positions 18 and 21 are replaced with arginines and the cysteine residues at positions 9 and 20 are replaced with alanines. “KCST-less” is a mutant encoding the combined substitutions of STS and KKCC.
Figure Legend Snippet: The YxY sequence in the BST2 cytoplasmic domain is required for signaling but dispensable for restriction. (A) HEK293T cells were transfected in wells of a 24-well plate with a plasmid encoding firefly luciferase under the control of a minimal promoter containing binding sites for NF-κB (200 ng) along with increasing amounts of a plasmid expressing BST2 (indicated); a plasmid expressing β-galactosidase (220 ng) was included as a normalization control. The next day, the cells were lysed and luciferase and β-galactosidase activities were measured by luminometry. Data are the ratio of relative light units (RLU) measured for luciferase activity to RLU measured for β-galactosidase activity and are the average of duplicates. (B) Restriction of HIV-1 virion release by BST2. HEK293T cells were transfected in wells of a 12-well plate with a plasmid encoding an HIV-1 provirus lacking vpu (1.6 μg) along with a plasmid encoding either no BST2, wild-type BST2, or the indicated mutants (30 ng). The next day, the supernatants were removed and the virions pelleted though a 20% sucrose cushion and then resuspended in the original culture volume before measurement of p24 Gag (capsid) concentration by ELISA. Data are the average of duplicates. (C) Cells from the experiment shown in panel B were analyzed by SDS-PAGE and immunoblotting for BST2 and p55 Gag precursor. In all panels, Y6/8A is a BST2 mutant in which the tyrosines at positions 6 and 8 are replaced with alanines, STS is a mutant in which the serine and threonine residues at positions 3 to 5 are replaced with alanines, and KKCC is a mutant in which the lysine residues at positions 18 and 21 are replaced with arginines and the cysteine residues at positions 9 and 20 are replaced with alanines. “KCST-less” is a mutant encoding the combined substitutions of STS and KKCC.

Techniques Used: Sequencing, Transfection, Plasmid Preparation, Luciferase, Binding Assay, Expressing, Activity Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, SDS Page, Mutagenesis

HIV-1 Vpu inhibits BST2-mediated signaling. (A) HEK293T cells were transfected in wells of a 24-well plate with a plasmid encoding firefly luciferase under the control of a minimal promoter containing binding sites for NF-κB, along with increasing amounts of a plasmid expressing BST2 and fixed amounts (65 ng) of plasmids expressing either Vpu, the indicated Vpu mutants, HLA-A2, or nothing (BST2 only). Vpu3A/F is the A10,14,18F mutant of the interactive face of the Vpu transmembrane helix, Vpu2/6 is the S52,56N mutant of the β-TrCP binding site, and Vpu3A/F,2/6 is a combination mutant. A plasmid expressing β-galactosidase was included as a normalization control. Data are the ratio of relative light units (RLU) measured for luciferase activity to RLU measured for β-galactosidase activity and are the average of duplicates. (B) Cells from parallel transfections set up as for panel A and containing 10 ng of BST2 expression plasmid were analyzed by SDS-PAGE and immunoblotting for BST2.
Figure Legend Snippet: HIV-1 Vpu inhibits BST2-mediated signaling. (A) HEK293T cells were transfected in wells of a 24-well plate with a plasmid encoding firefly luciferase under the control of a minimal promoter containing binding sites for NF-κB, along with increasing amounts of a plasmid expressing BST2 and fixed amounts (65 ng) of plasmids expressing either Vpu, the indicated Vpu mutants, HLA-A2, or nothing (BST2 only). Vpu3A/F is the A10,14,18F mutant of the interactive face of the Vpu transmembrane helix, Vpu2/6 is the S52,56N mutant of the β-TrCP binding site, and Vpu3A/F,2/6 is a combination mutant. A plasmid expressing β-galactosidase was included as a normalization control. Data are the ratio of relative light units (RLU) measured for luciferase activity to RLU measured for β-galactosidase activity and are the average of duplicates. (B) Cells from parallel transfections set up as for panel A and containing 10 ng of BST2 expression plasmid were analyzed by SDS-PAGE and immunoblotting for BST2.

Techniques Used: Transfection, Plasmid Preparation, Luciferase, Binding Assay, Expressing, Mutagenesis, Activity Assay, SDS Page

Pathways of BST2-mediated signaling. (A) Induction of NF-κB activity by BST2 is decreased by an inhibitor of TAK1 kinase and by intracellular chelation of calcium. Left panel, HEK293T cells were transfected in wells of a 24-well plate with a plasmid encoding firefly luciferase under the control of a minimal promoter containing binding sites for NF-κB (80 ng) along with increasing amounts of a plasmid expressing BST2; a plasmid expressing β-galactosidase (40 ng) was included as a normalization control. Immediately after the transfection, 1 μM (5 Z )-7-oxozeaenol (5OZ), a selective inhibitor of TAK1), 40 μM BAPTA-AM, a cell-permeative calcium chelator, or nothing was added. The next day, the cells were lysed, and luciferase and β-galactosidase activities were measured by luminometry. Data are the ratio of relative light units (RLU) measured for luciferase activity to RLU measured for β-galactosidase activity and are the average of duplicates. Right panel, fold induction of NF-κB activity in HEK293 cells transfected as described above but without a plasmid expressing BST2 and treated with 1 ng/ml TNF either alone or with 5OZ or BAPTA-AM. (B) Dominant-negative IκB blocks the induction of NF-κB activity by BST2. HEK293T cells were transfected and analyzed as for panel A, but the transfection mixtures included 200 ng of NF-κB-luc indicator, 220 ng of β-galactosidase expression plasmid, and, in addition to a dose range of plasmid expressing BST2, 20 ng of a plasmid expressing either wild-type IκB or dominant-negative IκB (DN-IκB). (C) BST2 coimmunoprecipitates with MyD88, TAB1, TAB2, and TAK1. HEK293T cells were transfected to express FLAG-tagged MyD88, TRAF2, TRAF3, TRAF6, TAB1, TAB2, or TAK1 and untagged BST2. The next day, the cells were lysed and immunoprecipitated with anti-FLAG conjugated beads. Immunoprecipitates (IP-FLAG) were analyzed for the FLAG-tagged proteins and BST2 as indicated. (D and E) Interaction with TAK1 and TAB1 requires the tyrosine residues at positions 6 and 8 but not the leucine residue at position 70 in BST2. HEK293T cells were transfected to express either FLAG-tagged TAK1 (D) or FLAG-tagged TAB1 (E) and untagged BST2 or the indicated mutants. The next day, the cells were lysed and immunoprecipitated with anti-FLAG conjugated beads. Lysates and immunoprecipitates were analyzed for the FLAG-tagged proteins and BST2 as indicated.
Figure Legend Snippet: Pathways of BST2-mediated signaling. (A) Induction of NF-κB activity by BST2 is decreased by an inhibitor of TAK1 kinase and by intracellular chelation of calcium. Left panel, HEK293T cells were transfected in wells of a 24-well plate with a plasmid encoding firefly luciferase under the control of a minimal promoter containing binding sites for NF-κB (80 ng) along with increasing amounts of a plasmid expressing BST2; a plasmid expressing β-galactosidase (40 ng) was included as a normalization control. Immediately after the transfection, 1 μM (5 Z )-7-oxozeaenol (5OZ), a selective inhibitor of TAK1), 40 μM BAPTA-AM, a cell-permeative calcium chelator, or nothing was added. The next day, the cells were lysed, and luciferase and β-galactosidase activities were measured by luminometry. Data are the ratio of relative light units (RLU) measured for luciferase activity to RLU measured for β-galactosidase activity and are the average of duplicates. Right panel, fold induction of NF-κB activity in HEK293 cells transfected as described above but without a plasmid expressing BST2 and treated with 1 ng/ml TNF either alone or with 5OZ or BAPTA-AM. (B) Dominant-negative IκB blocks the induction of NF-κB activity by BST2. HEK293T cells were transfected and analyzed as for panel A, but the transfection mixtures included 200 ng of NF-κB-luc indicator, 220 ng of β-galactosidase expression plasmid, and, in addition to a dose range of plasmid expressing BST2, 20 ng of a plasmid expressing either wild-type IκB or dominant-negative IκB (DN-IκB). (C) BST2 coimmunoprecipitates with MyD88, TAB1, TAB2, and TAK1. HEK293T cells were transfected to express FLAG-tagged MyD88, TRAF2, TRAF3, TRAF6, TAB1, TAB2, or TAK1 and untagged BST2. The next day, the cells were lysed and immunoprecipitated with anti-FLAG conjugated beads. Immunoprecipitates (IP-FLAG) were analyzed for the FLAG-tagged proteins and BST2 as indicated. (D and E) Interaction with TAK1 and TAB1 requires the tyrosine residues at positions 6 and 8 but not the leucine residue at position 70 in BST2. HEK293T cells were transfected to express either FLAG-tagged TAK1 (D) or FLAG-tagged TAB1 (E) and untagged BST2 or the indicated mutants. The next day, the cells were lysed and immunoprecipitated with anti-FLAG conjugated beads. Lysates and immunoprecipitates were analyzed for the FLAG-tagged proteins and BST2 as indicated.

Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Luciferase, Binding Assay, Expressing, Dominant Negative Mutation, Immunoprecipitation

6) Product Images from "MBDin, a Novel MBD2-Interacting Protein, Relieves MBD2 Repression Potential and Reactivates Transcription from Methylated Promoters"

Article Title: MBDin, a Novel MBD2-Interacting Protein, Relieves MBD2 Repression Potential and Reactivates Transcription from Methylated Promoters

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.5.1656-1665.2003

Mapping of MBD2 and MBD in binding region. (A) Schematic representation of structural and functional domains of MBD2 and MBD in proteins. For MBD2, the MBD is indicated as a black box; the putative coiled-coil domain is indicated as a gray box. For MBD in , the black box indicates the GTP-binding site, the dotted box indicates the region of homology with GTPases, the gray box indicates the NES motif, and the hatched box indicates the C-terminal region containing a cluster of acidic amino acid residues. (B) Mapping of the MBD in -binding region of MBD2 by yeast two-hybrid experiments. Full-length MBD2 and deletion mutants were fused to the Gal4 DBD (bait), and full-length MBD in was fused to the Gal4AD (prey). The interaction between the bait proteins and prey was tested on the basis of the His and Ade reporter gene activation and by quantitative liquid β-galactosidase assay. (C) Mapping of the MBD2-binding region of MBD in by yeast two-hybrid experiments. Full-length MBD in and mutants were fused to the Gal4 DNA activation domain, and full-length MBD2 was fused to the Gal4BD. The interaction between the bait proteins and prey was tested as described above.
Figure Legend Snippet: Mapping of MBD2 and MBD in binding region. (A) Schematic representation of structural and functional domains of MBD2 and MBD in proteins. For MBD2, the MBD is indicated as a black box; the putative coiled-coil domain is indicated as a gray box. For MBD in , the black box indicates the GTP-binding site, the dotted box indicates the region of homology with GTPases, the gray box indicates the NES motif, and the hatched box indicates the C-terminal region containing a cluster of acidic amino acid residues. (B) Mapping of the MBD in -binding region of MBD2 by yeast two-hybrid experiments. Full-length MBD2 and deletion mutants were fused to the Gal4 DBD (bait), and full-length MBD in was fused to the Gal4AD (prey). The interaction between the bait proteins and prey was tested on the basis of the His and Ade reporter gene activation and by quantitative liquid β-galactosidase assay. (C) Mapping of the MBD2-binding region of MBD in by yeast two-hybrid experiments. Full-length MBD in and mutants were fused to the Gal4 DNA activation domain, and full-length MBD2 was fused to the Gal4BD. The interaction between the bait proteins and prey was tested as described above.

Techniques Used: Binding Assay, Functional Assay, Activation Assay

Influence of MBD2 and MBD in overexpression on nonmethylated and methylated promoter activity. 293T cells were transfected with 5 μg of nonmethylated (A) or in vitro-methylated (B) pGAT1700 reporter plasmid. Cotransfected expression plasmids were pCMVβ (1 μg) and, where indicated, pMyc-MBD2 (3 μg) and pHA-MBD in in different amounts. The level of transcription is given as a percentage of the CAT activity seen in the presence of unmethylated pGAT1700 reporter plasmid alone. Percent relative activity of the promoters is expressed as the ratio of CAT to β-galactosidase activity. The results are averages of multiple experiments plus standard deviations. Each bar represents the mean of four independent transfections.
Figure Legend Snippet: Influence of MBD2 and MBD in overexpression on nonmethylated and methylated promoter activity. 293T cells were transfected with 5 μg of nonmethylated (A) or in vitro-methylated (B) pGAT1700 reporter plasmid. Cotransfected expression plasmids were pCMVβ (1 μg) and, where indicated, pMyc-MBD2 (3 μg) and pHA-MBD in in different amounts. The level of transcription is given as a percentage of the CAT activity seen in the presence of unmethylated pGAT1700 reporter plasmid alone. Percent relative activity of the promoters is expressed as the ratio of CAT to β-galactosidase activity. The results are averages of multiple experiments plus standard deviations. Each bar represents the mean of four independent transfections.

Techniques Used: Over Expression, Methylation, Activity Assay, Transfection, In Vitro, Plasmid Preparation, Expressing

7) Product Images from "Nrf2-Dependent Induction of NQO1 in Mouse Aortic Endothelial Cells Overexpressing Catalase"

Article Title: Nrf2-Dependent Induction of NQO1 in Mouse Aortic Endothelial Cells Overexpressing Catalase

Journal: Free radical biology & medicine

doi: 10.1016/j.freeradbiomed.2011.04.020

Functional analysis of the NQO1 promoter. NQO1 promoter-luciferase reporter plasmids were constructed and cotransfected into wild-type and hCat Tg MAECs with a β-galactosidase expression plasmid as described in Materials and Methods. (A) Schematic diagram depicting wild-type (P1), ARE-mutated (P2), and XRE-mutated (P3) NQO1 promoter-luciferase constructs. Luciferase activity was measured using a luminescence assay and expressed relative to the luminosity of β-galactosidase assay (B). Values represent the mean ± SEM of five independent experiments. * P
Figure Legend Snippet: Functional analysis of the NQO1 promoter. NQO1 promoter-luciferase reporter plasmids were constructed and cotransfected into wild-type and hCat Tg MAECs with a β-galactosidase expression plasmid as described in Materials and Methods. (A) Schematic diagram depicting wild-type (P1), ARE-mutated (P2), and XRE-mutated (P3) NQO1 promoter-luciferase constructs. Luciferase activity was measured using a luminescence assay and expressed relative to the luminosity of β-galactosidase assay (B). Values represent the mean ± SEM of five independent experiments. * P

Techniques Used: Functional Assay, Luciferase, Construct, Expressing, Plasmid Preparation, Activity Assay, Luminescence Assay

8) Product Images from "Targeting Estrogen-Related Receptor Alpha Inhibits Epithelial-to-Mesenchymal Transition and Stem Cell Properties of Ovarian Cancer Cells"

Article Title: Targeting Estrogen-Related Receptor Alpha Inhibits Epithelial-to-Mesenchymal Transition and Stem Cell Properties of Ovarian Cancer Cells

Journal: Molecular Therapy

doi: 10.1038/mt.2014.1

ERRα activates the promoter of Snail and attenuates its mRNA degradation. ( a ) OVCAR-3 cells were transiently transfected with 1.5 µg of the Snail promoter and 15 ng of β-galactosidase plasmid for 24 hours. Luciferase and
Figure Legend Snippet: ERRα activates the promoter of Snail and attenuates its mRNA degradation. ( a ) OVCAR-3 cells were transiently transfected with 1.5 µg of the Snail promoter and 15 ng of β-galactosidase plasmid for 24 hours. Luciferase and

Techniques Used: Transfection, Plasmid Preparation, Luciferase

Related Articles

Transfection:

Article Title: Glucocorticoids Regulate the Expression of the Human Osteoblastic Endothelin A Receptor Gene
Article Snippet: .. Before transfection, calculated for each well of a six-well plate, 10 μl Lipofection reagent was incubated for 10 min at room temperature with 1.5 μg of reporter gene construct, which contains the human ETRA 5′-flanking fragment (−3050 to +48) fused to luciferase gene, 0.5 μg glucocorticoid receptor expression plasmid when cotransfection experiments were performed (American Type Culture Collection, Rockville, MD), and 1.0 μg β-galactosidase expression plasmid (pSV-βgal; Promega Corp. ). ..

Article Title: Targeting Estrogen-Related Receptor Alpha Inhibits Epithelial-to-Mesenchymal Transition and Stem Cell Properties of Ovarian Cancer Cells
Article Snippet: .. Cells were transiently transfected with 1.5 µg of the 5′ promoter region of Snail (a kind gift of Dr. A. Garcia de Herreros, Universitat Pompeu Fabra, Barcelona, Spain) and cotransfected with 15 ng of β-galactosidase expression plasmid (pSV-β-gal; Promega, Madison, WI), using Lipofectamine 2000 reagent (Invitrogen). .. At 24 hours after transfection, luciferase activities were assayed using the Luciferase Reporter Assay kit according to the manufacturer's protocol (Promega). β-Galactosidase activity was used to normalize the transfection.

Article Title: MBDin, a Novel MBD2-Interacting Protein, Relieves MBD2 Repression Potential and Reactivates Transcription from Methylated Promoters
Article Snippet: .. For normalization of transfection efficiencies, a β-galactosidase expression plasmid, pSVβ-gal (Promega) (1 μg), was included in the cotransfection mixtures as an internal standard for transfection efficiency. .. Chloramphenicol acetyltransferase (CAT) assays were performed with different amounts of extracts to ensure linear conversion of the chloramphenicol with each extract, and results are presented as the means of at least three independent transfection experiments.

Article Title: TACC3 mediates the association of MBD2 with histone acetyltransferases and relieves transcriptional repression of methylated promoters
Article Snippet: .. The β-galactosidase expression plasmid, pSVβ−gal (Promega) (2 µg), was used as an internal control for transfection efficiency. .. Chloramphenicol acetyltransferase (CAT) assays were performed with different amounts of extracts to ensure linear conversion of the chloramphenicol with each extract and the results are presented as the means of at least three independent transfection experiments.

Luciferase:

Article Title: Glucocorticoids Regulate the Expression of the Human Osteoblastic Endothelin A Receptor Gene
Article Snippet: .. Before transfection, calculated for each well of a six-well plate, 10 μl Lipofection reagent was incubated for 10 min at room temperature with 1.5 μg of reporter gene construct, which contains the human ETRA 5′-flanking fragment (−3050 to +48) fused to luciferase gene, 0.5 μg glucocorticoid receptor expression plasmid when cotransfection experiments were performed (American Type Culture Collection, Rockville, MD), and 1.0 μg β-galactosidase expression plasmid (pSV-βgal; Promega Corp. ). ..

Expressing:

Article Title: Evidence against a Role for ?-Arrestin1 in STAT1 Dephosphorylation and the Inhibition of Interferon-? Signaling
Article Snippet: .. Plasmid pSV-βgal, a β-galactosidase expression plasmid, was purchased from Promega. .. Human (#407306) and mouse IFNγ (#407303) were obtained from Merck Millipore.

Article Title: Glucocorticoids Regulate the Expression of the Human Osteoblastic Endothelin A Receptor Gene
Article Snippet: .. Before transfection, calculated for each well of a six-well plate, 10 μl Lipofection reagent was incubated for 10 min at room temperature with 1.5 μg of reporter gene construct, which contains the human ETRA 5′-flanking fragment (−3050 to +48) fused to luciferase gene, 0.5 μg glucocorticoid receptor expression plasmid when cotransfection experiments were performed (American Type Culture Collection, Rockville, MD), and 1.0 μg β-galactosidase expression plasmid (pSV-βgal; Promega Corp. ). ..

Article Title: Nrf2-Dependent Induction of NQO1 in Mouse Aortic Endothelial Cells Overexpressing Catalase
Article Snippet: .. The recombinant plasmid constructs and a β-galactosidase expression plasmid, pCMV-SPORT-βgal (Promega) were co-transfected into MAECs using the Lipofectamine 2000 kit (Invitrogen) according to the manufacturer’s protocol. .. Briefly, MAECs grown in six-well plates to approximately 60-70% confluence were incubated with 2 ml/well serum-free DMEM containing 10 μl of Lipofectamine 2000, 0.5 μg of β-galactosidase expression plasmid, and 4 μg of pGL2 plasmid encoding either the wild-type or mutated NQO1 promoter fragments.

Article Title: Targeting Estrogen-Related Receptor Alpha Inhibits Epithelial-to-Mesenchymal Transition and Stem Cell Properties of Ovarian Cancer Cells
Article Snippet: .. Cells were transiently transfected with 1.5 µg of the 5′ promoter region of Snail (a kind gift of Dr. A. Garcia de Herreros, Universitat Pompeu Fabra, Barcelona, Spain) and cotransfected with 15 ng of β-galactosidase expression plasmid (pSV-β-gal; Promega, Madison, WI), using Lipofectamine 2000 reagent (Invitrogen). .. At 24 hours after transfection, luciferase activities were assayed using the Luciferase Reporter Assay kit according to the manufacturer's protocol (Promega). β-Galactosidase activity was used to normalize the transfection.

Article Title: MBDin, a Novel MBD2-Interacting Protein, Relieves MBD2 Repression Potential and Reactivates Transcription from Methylated Promoters
Article Snippet: .. For normalization of transfection efficiencies, a β-galactosidase expression plasmid, pSVβ-gal (Promega) (1 μg), was included in the cotransfection mixtures as an internal standard for transfection efficiency. .. Chloramphenicol acetyltransferase (CAT) assays were performed with different amounts of extracts to ensure linear conversion of the chloramphenicol with each extract, and results are presented as the means of at least three independent transfection experiments.

Article Title: Stimulation of NF-?B Activity by the HIV Restriction Factor BST2
Article Snippet: .. The NF-κB-luc reporter plasmid and the β-galactosidase expression plasmid were purchased from Promega. .. The ISRE-luc reporter plasmid, the IRF-3 5D expression plasmid, and plasmids expressing FLAG-tagged MyD88, TRAF3, TRAF6, or TAB2 were provided by Sumit Chanda.

Article Title: TACC3 mediates the association of MBD2 with histone acetyltransferases and relieves transcriptional repression of methylated promoters
Article Snippet: .. The β-galactosidase expression plasmid, pSVβ−gal (Promega) (2 µg), was used as an internal control for transfection efficiency. .. Chloramphenicol acetyltransferase (CAT) assays were performed with different amounts of extracts to ensure linear conversion of the chloramphenicol with each extract and the results are presented as the means of at least three independent transfection experiments.

Construct:

Article Title: Glucocorticoids Regulate the Expression of the Human Osteoblastic Endothelin A Receptor Gene
Article Snippet: .. Before transfection, calculated for each well of a six-well plate, 10 μl Lipofection reagent was incubated for 10 min at room temperature with 1.5 μg of reporter gene construct, which contains the human ETRA 5′-flanking fragment (−3050 to +48) fused to luciferase gene, 0.5 μg glucocorticoid receptor expression plasmid when cotransfection experiments were performed (American Type Culture Collection, Rockville, MD), and 1.0 μg β-galactosidase expression plasmid (pSV-βgal; Promega Corp. ). ..

Article Title: Nrf2-Dependent Induction of NQO1 in Mouse Aortic Endothelial Cells Overexpressing Catalase
Article Snippet: .. The recombinant plasmid constructs and a β-galactosidase expression plasmid, pCMV-SPORT-βgal (Promega) were co-transfected into MAECs using the Lipofectamine 2000 kit (Invitrogen) according to the manufacturer’s protocol. .. Briefly, MAECs grown in six-well plates to approximately 60-70% confluence were incubated with 2 ml/well serum-free DMEM containing 10 μl of Lipofectamine 2000, 0.5 μg of β-galactosidase expression plasmid, and 4 μg of pGL2 plasmid encoding either the wild-type or mutated NQO1 promoter fragments.

Article Title: A Novel Function of Apolipoprotein E: Upregulation of ATP-Binding Cassette Transporter A1 Expression
Article Snippet: .. The recombinant plasmid construct (or empty pGL-2 vector) and a β-galactosidase expression plasmid (pCMV-SPORT-βgal, Promega) were co-transfected into RAW264.7 cells using a Nucleofector II as described previously . .. The transfected cells were treated with 20 µg/ml of E+ /B, E− /B or E3/B lipoproteins or culture medium alone for 4 hrs, and lysed with 100 µl of lysis buffer provided by the luciferase assay kit or the galactosidase assay kit (Galacto-Star™ ß-Galactosidase Reporter Gene Assay System).

Incubation:

Article Title: Glucocorticoids Regulate the Expression of the Human Osteoblastic Endothelin A Receptor Gene
Article Snippet: .. Before transfection, calculated for each well of a six-well plate, 10 μl Lipofection reagent was incubated for 10 min at room temperature with 1.5 μg of reporter gene construct, which contains the human ETRA 5′-flanking fragment (−3050 to +48) fused to luciferase gene, 0.5 μg glucocorticoid receptor expression plasmid when cotransfection experiments were performed (American Type Culture Collection, Rockville, MD), and 1.0 μg β-galactosidase expression plasmid (pSV-βgal; Promega Corp. ). ..

Cotransfection:

Article Title: Glucocorticoids Regulate the Expression of the Human Osteoblastic Endothelin A Receptor Gene
Article Snippet: .. Before transfection, calculated for each well of a six-well plate, 10 μl Lipofection reagent was incubated for 10 min at room temperature with 1.5 μg of reporter gene construct, which contains the human ETRA 5′-flanking fragment (−3050 to +48) fused to luciferase gene, 0.5 μg glucocorticoid receptor expression plasmid when cotransfection experiments were performed (American Type Culture Collection, Rockville, MD), and 1.0 μg β-galactosidase expression plasmid (pSV-βgal; Promega Corp. ). ..

Article Title: MBDin, a Novel MBD2-Interacting Protein, Relieves MBD2 Repression Potential and Reactivates Transcription from Methylated Promoters
Article Snippet: .. For normalization of transfection efficiencies, a β-galactosidase expression plasmid, pSVβ-gal (Promega) (1 μg), was included in the cotransfection mixtures as an internal standard for transfection efficiency. .. Chloramphenicol acetyltransferase (CAT) assays were performed with different amounts of extracts to ensure linear conversion of the chloramphenicol with each extract, and results are presented as the means of at least three independent transfection experiments.

Recombinant:

Article Title: Nrf2-Dependent Induction of NQO1 in Mouse Aortic Endothelial Cells Overexpressing Catalase
Article Snippet: .. The recombinant plasmid constructs and a β-galactosidase expression plasmid, pCMV-SPORT-βgal (Promega) were co-transfected into MAECs using the Lipofectamine 2000 kit (Invitrogen) according to the manufacturer’s protocol. .. Briefly, MAECs grown in six-well plates to approximately 60-70% confluence were incubated with 2 ml/well serum-free DMEM containing 10 μl of Lipofectamine 2000, 0.5 μg of β-galactosidase expression plasmid, and 4 μg of pGL2 plasmid encoding either the wild-type or mutated NQO1 promoter fragments.

Article Title: A Novel Function of Apolipoprotein E: Upregulation of ATP-Binding Cassette Transporter A1 Expression
Article Snippet: .. The recombinant plasmid construct (or empty pGL-2 vector) and a β-galactosidase expression plasmid (pCMV-SPORT-βgal, Promega) were co-transfected into RAW264.7 cells using a Nucleofector II as described previously . .. The transfected cells were treated with 20 µg/ml of E+ /B, E− /B or E3/B lipoproteins or culture medium alone for 4 hrs, and lysed with 100 µl of lysis buffer provided by the luciferase assay kit or the galactosidase assay kit (Galacto-Star™ ß-Galactosidase Reporter Gene Assay System).

Plasmid Preparation:

Article Title: Evidence against a Role for ?-Arrestin1 in STAT1 Dephosphorylation and the Inhibition of Interferon-? Signaling
Article Snippet: .. Plasmid pSV-βgal, a β-galactosidase expression plasmid, was purchased from Promega. .. Human (#407306) and mouse IFNγ (#407303) were obtained from Merck Millipore.

Article Title: Glucocorticoids Regulate the Expression of the Human Osteoblastic Endothelin A Receptor Gene
Article Snippet: .. Before transfection, calculated for each well of a six-well plate, 10 μl Lipofection reagent was incubated for 10 min at room temperature with 1.5 μg of reporter gene construct, which contains the human ETRA 5′-flanking fragment (−3050 to +48) fused to luciferase gene, 0.5 μg glucocorticoid receptor expression plasmid when cotransfection experiments were performed (American Type Culture Collection, Rockville, MD), and 1.0 μg β-galactosidase expression plasmid (pSV-βgal; Promega Corp. ). ..

Article Title: Nrf2-Dependent Induction of NQO1 in Mouse Aortic Endothelial Cells Overexpressing Catalase
Article Snippet: .. The recombinant plasmid constructs and a β-galactosidase expression plasmid, pCMV-SPORT-βgal (Promega) were co-transfected into MAECs using the Lipofectamine 2000 kit (Invitrogen) according to the manufacturer’s protocol. .. Briefly, MAECs grown in six-well plates to approximately 60-70% confluence were incubated with 2 ml/well serum-free DMEM containing 10 μl of Lipofectamine 2000, 0.5 μg of β-galactosidase expression plasmid, and 4 μg of pGL2 plasmid encoding either the wild-type or mutated NQO1 promoter fragments.

Article Title: Targeting Estrogen-Related Receptor Alpha Inhibits Epithelial-to-Mesenchymal Transition and Stem Cell Properties of Ovarian Cancer Cells
Article Snippet: .. Cells were transiently transfected with 1.5 µg of the 5′ promoter region of Snail (a kind gift of Dr. A. Garcia de Herreros, Universitat Pompeu Fabra, Barcelona, Spain) and cotransfected with 15 ng of β-galactosidase expression plasmid (pSV-β-gal; Promega, Madison, WI), using Lipofectamine 2000 reagent (Invitrogen). .. At 24 hours after transfection, luciferase activities were assayed using the Luciferase Reporter Assay kit according to the manufacturer's protocol (Promega). β-Galactosidase activity was used to normalize the transfection.

Article Title: MBDin, a Novel MBD2-Interacting Protein, Relieves MBD2 Repression Potential and Reactivates Transcription from Methylated Promoters
Article Snippet: .. For normalization of transfection efficiencies, a β-galactosidase expression plasmid, pSVβ-gal (Promega) (1 μg), was included in the cotransfection mixtures as an internal standard for transfection efficiency. .. Chloramphenicol acetyltransferase (CAT) assays were performed with different amounts of extracts to ensure linear conversion of the chloramphenicol with each extract, and results are presented as the means of at least three independent transfection experiments.

Article Title: Stimulation of NF-?B Activity by the HIV Restriction Factor BST2
Article Snippet: .. The NF-κB-luc reporter plasmid and the β-galactosidase expression plasmid were purchased from Promega. .. The ISRE-luc reporter plasmid, the IRF-3 5D expression plasmid, and plasmids expressing FLAG-tagged MyD88, TRAF3, TRAF6, or TAB2 were provided by Sumit Chanda.

Article Title: TACC3 mediates the association of MBD2 with histone acetyltransferases and relieves transcriptional repression of methylated promoters
Article Snippet: .. The β-galactosidase expression plasmid, pSVβ−gal (Promega) (2 µg), was used as an internal control for transfection efficiency. .. Chloramphenicol acetyltransferase (CAT) assays were performed with different amounts of extracts to ensure linear conversion of the chloramphenicol with each extract and the results are presented as the means of at least three independent transfection experiments.

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    Promega β galactosidase expression plasmid
    Influence of MBD2 and TACC3 over-expression on activity of methylated transfected or endogenous galectin-1 promoter activity. ( A ) 293T cells were transfected with 3 µg of unmethylated (gray bar) or in vitro methylated (black bars) pGAT1700 reporter plasmid. Cotransfected expression plasmids were pSVβ (2 µg) and, where indicated, pMyc-MBD2 (2 µg) and pHA-TACC3 at different amounts. The level of transcription is given as a percentage of CAT activity seen in the presence of unmethylated pGAT1700 reporter plasmid alone (set as 100%). Percent relative activity of the promoters is expressed as the ratio of CAT to <t>β-galactosidase</t> activity. The results are presented as an average of multiple experiments +/− SD. Each histogram bar represents the mean of at least four independent transfections. ( B ). Quantitative RT–PCR assays of endogenous galectin-1 mRNAs were performed on PC Cl3 cells untreated or treated with the indicated concentrations of 5-aza-2′deoxycytidine (Aza) or Trycostatin A (TSA) for 36 h. Where indicated (TACC3), cells were transfected with pHA-TACC3 expression vector. mRNA expression levels are shown as fold-induction compared with the PC Cl3 value, arbitrarily set as 1. The results are presented as an average of at least three independent experiments +/− SD.
    β Galactosidase Expression Plasmid, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 20 article reviews
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    β galactosidase expression plasmid - by Bioz Stars, 2020-09
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    92
    Promega β galactosidase expression plasmid prl sv40
    Downregulation of DNMT1 inhibits the Wnt signaling pathway. (A) Relative luciferase activity representing β-catenin/TCF-dependent transcription in untreated 95Dcells (control) and 95D cells transfected with NC or DNMT1 siRNA. Cells were transfected with TOPflash or FOPflash along with <t>pRL-SV40.</t> FOPflash served as a specificity control for TOPflash activity. The luciferase activity was normalized against Renilla activity. Experiments were repeated three times. (B) Western blot analysis of cytosolic and nuclear β-catenin expression in untreated 95D cells (control) and 95D cells transfected with NC or DNMT1 siRNA. (C) Western blot analysis of the expression of the β-catenin target cyclin D1 in untreated 95D cells (control) and 95D cells transfected with NC or DNMT1 siRNA. GAPDH served as a loading control. **P
    β Galactosidase Expression Plasmid Prl Sv40, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β galactosidase expression plasmid prl sv40/product/Promega
    Average 92 stars, based on 2 article reviews
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    Influence of MBD2 and TACC3 over-expression on activity of methylated transfected or endogenous galectin-1 promoter activity. ( A ) 293T cells were transfected with 3 µg of unmethylated (gray bar) or in vitro methylated (black bars) pGAT1700 reporter plasmid. Cotransfected expression plasmids were pSVβ (2 µg) and, where indicated, pMyc-MBD2 (2 µg) and pHA-TACC3 at different amounts. The level of transcription is given as a percentage of CAT activity seen in the presence of unmethylated pGAT1700 reporter plasmid alone (set as 100%). Percent relative activity of the promoters is expressed as the ratio of CAT to β-galactosidase activity. The results are presented as an average of multiple experiments +/− SD. Each histogram bar represents the mean of at least four independent transfections. ( B ). Quantitative RT–PCR assays of endogenous galectin-1 mRNAs were performed on PC Cl3 cells untreated or treated with the indicated concentrations of 5-aza-2′deoxycytidine (Aza) or Trycostatin A (TSA) for 36 h. Where indicated (TACC3), cells were transfected with pHA-TACC3 expression vector. mRNA expression levels are shown as fold-induction compared with the PC Cl3 value, arbitrarily set as 1. The results are presented as an average of at least three independent experiments +/− SD.

    Journal: Nucleic Acids Research

    Article Title: TACC3 mediates the association of MBD2 with histone acetyltransferases and relieves transcriptional repression of methylated promoters

    doi: 10.1093/nar/gkj400

    Figure Lengend Snippet: Influence of MBD2 and TACC3 over-expression on activity of methylated transfected or endogenous galectin-1 promoter activity. ( A ) 293T cells were transfected with 3 µg of unmethylated (gray bar) or in vitro methylated (black bars) pGAT1700 reporter plasmid. Cotransfected expression plasmids were pSVβ (2 µg) and, where indicated, pMyc-MBD2 (2 µg) and pHA-TACC3 at different amounts. The level of transcription is given as a percentage of CAT activity seen in the presence of unmethylated pGAT1700 reporter plasmid alone (set as 100%). Percent relative activity of the promoters is expressed as the ratio of CAT to β-galactosidase activity. The results are presented as an average of multiple experiments +/− SD. Each histogram bar represents the mean of at least four independent transfections. ( B ). Quantitative RT–PCR assays of endogenous galectin-1 mRNAs were performed on PC Cl3 cells untreated or treated with the indicated concentrations of 5-aza-2′deoxycytidine (Aza) or Trycostatin A (TSA) for 36 h. Where indicated (TACC3), cells were transfected with pHA-TACC3 expression vector. mRNA expression levels are shown as fold-induction compared with the PC Cl3 value, arbitrarily set as 1. The results are presented as an average of at least three independent experiments +/− SD.

    Article Snippet: The β-galactosidase expression plasmid, pSVβ−gal (Promega) (2 µg), was used as an internal control for transfection efficiency.

    Techniques: Over Expression, Activity Assay, Methylation, Transfection, In Vitro, Plasmid Preparation, Expressing, Quantitative RT-PCR

    Definition of MBD2 and TACC3 binding region in yeast. ( A ) Schematic representation of structural and functional domains of MBD2 and TACC3 proteins. For MBD2, the methyl-binding-domain (MBD) is indicated as a black box; the putative coiled–coil domain is indicated as a gray box. For TACC3, the striped box contains a conserved N-terminal region and the TACC domain is indicated as a gray box. TACC3-mir (minimal interacting region) represents the minimal TACC3 region interacting with MBD2 in yeast. ( B ) Mapping of the TACC3-binding region of MBD2 by yeast two-hybrid experiments. Full length and deletion mutants of MBD2 were fused to GAL4 DNA binding domain (bait) and the TACC3-mir (amino acids from 571 to 837) was fused to the GAL4 activation domain (pGAD-F3 plasmid clone). The interaction between the bait proteins and prey was tested on the basis of the His and Ade reporter gene activation and by quantitative liquid β-galactosidase assay.

    Journal: Nucleic Acids Research

    Article Title: TACC3 mediates the association of MBD2 with histone acetyltransferases and relieves transcriptional repression of methylated promoters

    doi: 10.1093/nar/gkj400

    Figure Lengend Snippet: Definition of MBD2 and TACC3 binding region in yeast. ( A ) Schematic representation of structural and functional domains of MBD2 and TACC3 proteins. For MBD2, the methyl-binding-domain (MBD) is indicated as a black box; the putative coiled–coil domain is indicated as a gray box. For TACC3, the striped box contains a conserved N-terminal region and the TACC domain is indicated as a gray box. TACC3-mir (minimal interacting region) represents the minimal TACC3 region interacting with MBD2 in yeast. ( B ) Mapping of the TACC3-binding region of MBD2 by yeast two-hybrid experiments. Full length and deletion mutants of MBD2 were fused to GAL4 DNA binding domain (bait) and the TACC3-mir (amino acids from 571 to 837) was fused to the GAL4 activation domain (pGAD-F3 plasmid clone). The interaction between the bait proteins and prey was tested on the basis of the His and Ade reporter gene activation and by quantitative liquid β-galactosidase assay.

    Article Snippet: The β-galactosidase expression plasmid, pSVβ−gal (Promega) (2 µg), was used as an internal control for transfection efficiency.

    Techniques: Binding Assay, Functional Assay, Activation Assay, Plasmid Preparation

    ( A ) Schematic demonstration of the chimeric construct of the 5′-flanking region of the ETR A gene fused to luciferase gene in pGL3 Basic vector. ( B ) Reporter gene activity was assayed in HOC cotransfected with reporter gene construct and glucocorticoid receptor expression plasmid and β-galactosidase expression plasmid, treated for 24 h in parallel with 100 nM dexamethasone and control medium, and expressed in corrected luciferase activity ± SE.

    Journal: The Journal of Experimental Medicine

    Article Title: Glucocorticoids Regulate the Expression of the Human Osteoblastic Endothelin A Receptor Gene

    doi:

    Figure Lengend Snippet: ( A ) Schematic demonstration of the chimeric construct of the 5′-flanking region of the ETR A gene fused to luciferase gene in pGL3 Basic vector. ( B ) Reporter gene activity was assayed in HOC cotransfected with reporter gene construct and glucocorticoid receptor expression plasmid and β-galactosidase expression plasmid, treated for 24 h in parallel with 100 nM dexamethasone and control medium, and expressed in corrected luciferase activity ± SE.

    Article Snippet: Before transfection, calculated for each well of a six-well plate, 10 μl Lipofection reagent was incubated for 10 min at room temperature with 1.5 μg of reporter gene construct, which contains the human ETRA 5′-flanking fragment (−3050 to +48) fused to luciferase gene, 0.5 μg glucocorticoid receptor expression plasmid when cotransfection experiments were performed (American Type Culture Collection, Rockville, MD), and 1.0 μg β-galactosidase expression plasmid (pSV-βgal; Promega Corp. ).

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

    Cotransfection of β-Arrestin1 Has Nonspecific Inhibitory Effects on Gene Transcription (A and B) Interferon-γ-induced luciferase reporter activity in untreated and IFNγ-treated HeLa cells using increasing amounts of two β-arrestin1 constructs as indicated. Error bars represent means ± standard deviation for one representative experiment done in triplicates. The respective values for “fold induction” (induced/uninduced) are shown in the panels on the right. (C) Immunoblot showing the effects of FLAG-tagged β-arrestin1 on coexpressed β-galactosidase in HeLa cells. After western blotting, the nitrocellulose membrane was cut horizontally at the ∼80 kDa position. The upper membrane was probed with antibody against β-galactosidase; the other was consecutively probed with anti-FLAG-tag and anti-β-actin. (D and E) Interferon-γ-induced luciferase reporter activity in untreated and IFNγ-treated HeLa cells. The cells were transfected with plasmid pBS (control) or the expression plasmid for GFP-tagged β-arrestin1, and in addition with different ratios of plasmids encoding β-galactosidase and the luciferase reporter gene as indicated. Error bars represent means ± standard deviation of one experiment done in triplicate. The respective values for “fold induction” (induced/uninduced) are shown in (E).

    Journal: Molecular Cell

    Article Title: Evidence against a Role for ?-Arrestin1 in STAT1 Dephosphorylation and the Inhibition of Interferon-? Signaling

    doi: 10.1016/j.molcel.2013.02.024

    Figure Lengend Snippet: Cotransfection of β-Arrestin1 Has Nonspecific Inhibitory Effects on Gene Transcription (A and B) Interferon-γ-induced luciferase reporter activity in untreated and IFNγ-treated HeLa cells using increasing amounts of two β-arrestin1 constructs as indicated. Error bars represent means ± standard deviation for one representative experiment done in triplicates. The respective values for “fold induction” (induced/uninduced) are shown in the panels on the right. (C) Immunoblot showing the effects of FLAG-tagged β-arrestin1 on coexpressed β-galactosidase in HeLa cells. After western blotting, the nitrocellulose membrane was cut horizontally at the ∼80 kDa position. The upper membrane was probed with antibody against β-galactosidase; the other was consecutively probed with anti-FLAG-tag and anti-β-actin. (D and E) Interferon-γ-induced luciferase reporter activity in untreated and IFNγ-treated HeLa cells. The cells were transfected with plasmid pBS (control) or the expression plasmid for GFP-tagged β-arrestin1, and in addition with different ratios of plasmids encoding β-galactosidase and the luciferase reporter gene as indicated. Error bars represent means ± standard deviation of one experiment done in triplicate. The respective values for “fold induction” (induced/uninduced) are shown in (E).

    Article Snippet: Plasmid pSV-βgal, a β-galactosidase expression plasmid, was purchased from Promega.

    Techniques: Cotransfection, Luciferase, Activity Assay, Construct, Standard Deviation, Western Blot, FLAG-tag, Transfection, Plasmid Preparation, Expressing

    β-Arrestin1 Does Not Affect STAT1 Phosphorylation (A) Immunoblot showing a time course of STAT1 phosphorylation in IFNγ-treated HeLa cells that transiently express β-arrestin1 or β-galactosidase as indicated. Cells were left untreated or treated with IFNγ for 60 min, followed by incubation for 0–240 min in growth medium (GM) without interferon. Probing was done first with anti-Tyr701-phoshorylated STAT1 antibody, then with a mixture of anti-human STAT1 and anti-β-actin antibody, followed by a mixture of anti-GFP and anti-FLAG-tag, and finally with anti-β-galactosidase. Molecular weight marker positions are indicated. The diagram combines the results of this and another experiment. It shows specific Tyr701-phosphorylation of STAT1 under the different experimental conditions (specific Tyr701 phosphorylation after 60 min IFNγ was set as 100). Results were calculated as the mean ± standard deviation. (B) Shown are immunoblot analyses of wild-type (WT) and β-arrestin1-deficient MEFs using anti-β-arrestin1 antibody. Reprobing was done with anti-β-actin. Positions of molecular weight markers and β-arrestin 1 are indicated. (C) Top, immunoblot analyses of STAT1 dephosphorylation in WT and mutant MEFs. Bottom, diagram combining the results of this and two additional experiments giving specific Tyr701 phosphorylation of STAT1. The value for 60 min IFNγ was set as 100. Results were calculated as the mean ± standard deviation.

    Journal: Molecular Cell

    Article Title: Evidence against a Role for ?-Arrestin1 in STAT1 Dephosphorylation and the Inhibition of Interferon-? Signaling

    doi: 10.1016/j.molcel.2013.02.024

    Figure Lengend Snippet: β-Arrestin1 Does Not Affect STAT1 Phosphorylation (A) Immunoblot showing a time course of STAT1 phosphorylation in IFNγ-treated HeLa cells that transiently express β-arrestin1 or β-galactosidase as indicated. Cells were left untreated or treated with IFNγ for 60 min, followed by incubation for 0–240 min in growth medium (GM) without interferon. Probing was done first with anti-Tyr701-phoshorylated STAT1 antibody, then with a mixture of anti-human STAT1 and anti-β-actin antibody, followed by a mixture of anti-GFP and anti-FLAG-tag, and finally with anti-β-galactosidase. Molecular weight marker positions are indicated. The diagram combines the results of this and another experiment. It shows specific Tyr701-phosphorylation of STAT1 under the different experimental conditions (specific Tyr701 phosphorylation after 60 min IFNγ was set as 100). Results were calculated as the mean ± standard deviation. (B) Shown are immunoblot analyses of wild-type (WT) and β-arrestin1-deficient MEFs using anti-β-arrestin1 antibody. Reprobing was done with anti-β-actin. Positions of molecular weight markers and β-arrestin 1 are indicated. (C) Top, immunoblot analyses of STAT1 dephosphorylation in WT and mutant MEFs. Bottom, diagram combining the results of this and two additional experiments giving specific Tyr701 phosphorylation of STAT1. The value for 60 min IFNγ was set as 100. Results were calculated as the mean ± standard deviation.

    Article Snippet: Plasmid pSV-βgal, a β-galactosidase expression plasmid, was purchased from Promega.

    Techniques: Incubation, FLAG-tag, Molecular Weight, Marker, Standard Deviation, De-Phosphorylation Assay, Mutagenesis

    Downregulation of DNMT1 inhibits the Wnt signaling pathway. (A) Relative luciferase activity representing β-catenin/TCF-dependent transcription in untreated 95Dcells (control) and 95D cells transfected with NC or DNMT1 siRNA. Cells were transfected with TOPflash or FOPflash along with pRL-SV40. FOPflash served as a specificity control for TOPflash activity. The luciferase activity was normalized against Renilla activity. Experiments were repeated three times. (B) Western blot analysis of cytosolic and nuclear β-catenin expression in untreated 95D cells (control) and 95D cells transfected with NC or DNMT1 siRNA. (C) Western blot analysis of the expression of the β-catenin target cyclin D1 in untreated 95D cells (control) and 95D cells transfected with NC or DNMT1 siRNA. GAPDH served as a loading control. **P

    Journal: Oncology Letters

    Article Title: Inhibition of DNA methyltransferase 1 by RNA interference reverses epithelial-mesenchymal transition in highly metastatic 95D lung cancer cells by inhibiting the Wnt signaling pathway

    doi: 10.3892/ol.2018.8449

    Figure Lengend Snippet: Downregulation of DNMT1 inhibits the Wnt signaling pathway. (A) Relative luciferase activity representing β-catenin/TCF-dependent transcription in untreated 95Dcells (control) and 95D cells transfected with NC or DNMT1 siRNA. Cells were transfected with TOPflash or FOPflash along with pRL-SV40. FOPflash served as a specificity control for TOPflash activity. The luciferase activity was normalized against Renilla activity. Experiments were repeated three times. (B) Western blot analysis of cytosolic and nuclear β-catenin expression in untreated 95D cells (control) and 95D cells transfected with NC or DNMT1 siRNA. (C) Western blot analysis of the expression of the β-catenin target cyclin D1 in untreated 95D cells (control) and 95D cells transfected with NC or DNMT1 siRNA. GAPDH served as a loading control. **P

    Article Snippet: 95D cells transfected with NC or DNMT1 siRNA were co-transfected with TOPflash or FOPflash plasmids (Upstate Biotechnology, Inc., Lake Placid, NY, USA), along with β-galactosidase expression plasmid pRL-SV40 (Promega Corporation, Madison, WI, USA), using Lipofectamine® 2000 reagent.

    Techniques: Luciferase, Activity Assay, Transfection, Western Blot, Expressing