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( A ) M2H assays were performed in HEK293 cells to determine interactions between <t>VP16-ERα</t> and SIRT1, Sir2 and Sir-2.1 tethered to Gal4DBD. ( B ) SIRT1-PGC-1α transcriptional complexes strongly interact with ERα and ERβ and overide Gal4 promoter repression by SIRT1 alone. M2H assay was repeated with Gal4DBD-SIRT1 and Gal4DBD-PGC-1α, and ERα or ERβ fused to VP16 activation domain. ( C ) M2H assay for interaction between the STACs-AD and VP16-ERα or VP16-ERβ. Gal4DBD-STACs-AD Wt or its NR-box mutant (ΔNRB) were cotransfected with the receptors as indicated. Schematic shows locations of Wt and mutant NR-boxes within the STACs-AD. Statistical significance of differences in interaction (B) or gene expression in response to DMSO or E2 (A–C) are shown with P values: * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001; differences are not significant where P values are not shown. 5x Gal4-luc and β-gal activities were determined as described and ER interaction with Gal4DBD was set as reference point. Data were plotted in duplicates as means ± S.E.M and are representative of three independent experiments.

Vp16 Er Alpha, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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vp16 er alpha - by Bioz Stars, 2026-05

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1) Product Images from "A conserved mechanism of sirtuin signalling through steroid hormone receptors"

Article Title: A conserved mechanism of sirtuin signalling through steroid hormone receptors

Journal: Bioscience Reports

doi: 10.1042/BSR20193535

( A ) M2H assays were performed in HEK293 cells to determine interactions between VP16-ERα and SIRT1, Sir2 and Sir-2.1 tethered to Gal4DBD. ( B ) SIRT1-PGC-1α transcriptional complexes strongly interact with ERα and ERβ and overide Gal4 promoter repression by SIRT1 alone. M2H assay was repeated with Gal4DBD-SIRT1 and Gal4DBD-PGC-1α, and ERα or ERβ fused to VP16 activation domain. ( C ) M2H assay for interaction between the STACs-AD and VP16-ERα or VP16-ERβ. Gal4DBD-STACs-AD Wt or its NR-box mutant (ΔNRB) were cotransfected with the receptors as indicated. Schematic shows locations of Wt and mutant NR-boxes within the STACs-AD. Statistical significance of differences in interaction (B) or gene expression in response to DMSO or E2 (A–C) are shown with P values: * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001; differences are not significant where P values are not shown. 5x Gal4-luc and β-gal activities were determined as described and ER interaction with Gal4DBD was set as reference point. Data were plotted in duplicates as means ± S.E.M and are representative of three independent experiments.

Figure Legend Snippet: ( A ) M2H assays were performed in HEK293 cells to determine interactions between VP16-ERα and SIRT1, Sir2 and Sir-2.1 tethered to Gal4DBD. ( B ) SIRT1-PGC-1α transcriptional complexes strongly interact with ERα and ERβ and overide Gal4 promoter repression by SIRT1 alone. M2H assay was repeated with Gal4DBD-SIRT1 and Gal4DBD-PGC-1α, and ERα or ERβ fused to VP16 activation domain. ( C ) M2H assay for interaction between the STACs-AD and VP16-ERα or VP16-ERβ. Gal4DBD-STACs-AD Wt or its NR-box mutant (ΔNRB) were cotransfected with the receptors as indicated. Schematic shows locations of Wt and mutant NR-boxes within the STACs-AD. Statistical significance of differences in interaction (B) or gene expression in response to DMSO or E2 (A–C) are shown with P values: * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001; differences are not significant where P values are not shown. 5x Gal4-luc and β-gal activities were determined as described and ER interaction with Gal4DBD was set as reference point. Data were plotted in duplicates as means ± S.E.M and are representative of three independent experiments.

Techniques Used: Activation Assay, Mutagenesis, Expressing

Increasing doses (50, 100 and 200 ng) of Gal4DBD fusion peptides of wild-type (NRBWt) and mutant (NRBMt) SIRT1 NR-boxes were tested in M2H assays in HEK293 cells for interaction with ( A ) VP16-ERα and ( B ) VP16-ERβ; the reporter gene was 5x Gal4-luc. Luciferase expression was normalized to β-gal activity. Graphs were plotted by setting the activity of Gal4DBD interaction with the ERs as reference. P values show the significance of differences in ER interaction, comparing NRBWt with NRBMt; only P values ≤ 0.05 are significant. ( C–F ) Modified M2H assay of SIRT1 NR-box peptide binding specificity by competitive inhibition with wild-type (C,E) and mutant (D,F) NR-box peptides. Interactions were determined through dose-dependent repression of ERα (C,D) and ERβ (E,F) signalling; antagonism was observed with wild-type (C,E) but not with mutant (D,F) NR-box peptides. For panels (C–F), MCF-7 cells were cotransfected with VP16-ERα or VP16-ERβ, the ER reporter gene instead of 5x Gal4-luc, and increasing amounts (50, 100 and 200 ng) of Gal4DBD SIRT1 NR-box peptides. Cells were treated with DMSO or 100 nM E2 and ER reporter gene expression was normalized to β-gal internal control. Fold activation was determined from the activity of Gal4DBD (negative control) set at a value of 1. P values show significant ( P ≤ 0.05) differences between DMSO and E2-treated cells. Data are representative of three independent assays.

Figure Legend Snippet: Increasing doses (50, 100 and 200 ng) of Gal4DBD fusion peptides of wild-type (NRBWt) and mutant (NRBMt) SIRT1 NR-boxes were tested in M2H assays in HEK293 cells for interaction with ( A ) VP16-ERα and ( B ) VP16-ERβ; the reporter gene was 5x Gal4-luc. Luciferase expression was normalized to β-gal activity. Graphs were plotted by setting the activity of Gal4DBD interaction with the ERs as reference. P values show the significance of differences in ER interaction, comparing NRBWt with NRBMt; only P values ≤ 0.05 are significant. ( C–F ) Modified M2H assay of SIRT1 NR-box peptide binding specificity by competitive inhibition with wild-type (C,E) and mutant (D,F) NR-box peptides. Interactions were determined through dose-dependent repression of ERα (C,D) and ERβ (E,F) signalling; antagonism was observed with wild-type (C,E) but not with mutant (D,F) NR-box peptides. For panels (C–F), MCF-7 cells were cotransfected with VP16-ERα or VP16-ERβ, the ER reporter gene instead of 5x Gal4-luc, and increasing amounts (50, 100 and 200 ng) of Gal4DBD SIRT1 NR-box peptides. Cells were treated with DMSO or 100 nM E2 and ER reporter gene expression was normalized to β-gal internal control. Fold activation was determined from the activity of Gal4DBD (negative control) set at a value of 1. P values show significant ( P ≤ 0.05) differences between DMSO and E2-treated cells. Data are representative of three independent assays.

Techniques Used: Mutagenesis, Luciferase, Expressing, Activity Assay, Modification, Binding Assay, Inhibition, Activation Assay, Negative Control

( A ) SIRT1 elicits ER subtype-selectivity in liver cells, coactivating ERα but not ERβ; the NR-box is required for ligand-dependent coactivation. Wild-type SIRT1 or its NR-box mutant SIRT1ΔNRB and the STACs-AD expression vectors were cotransfected into Hep3B cells with VP16-ERα or VP16-ERβ and the ER reporter gene 3xERRE.EREluc. ( B ) Sirtuins are veritable ER coactivators. Hep3B cells were cotransfected with 3xERRE.EREluc and SIRT1, Sir2 and Sir-2.1, with or without ERα. ( C ) ER subtype selectivity by SIRT1 is similar to NcoA3/SRC-3. ( D ) SIRT1 and PGC-1α are independent veritable ER coactivators. ( E ) Comparision of ERα coactivation by the sirtuins NcoA3/SRC-3 and PGC-1α. As in ( D ) note the strong intrinsic transcriptional activity of PGC-1α compared with the sirtuins and NcoA3/SRC-3. ( F ) Sir-2.1 and Sir2 but not SIRT1 show relaxed specificity towards ERβ. In (A–F) Hep3B cells were cotransfected with sirtuins and the ER reporter (3xERRE.EREluc) alone and together with ERα or ERβ. Ligand-independent coactivation was determined by incubating cells with DMSO while ligand-dependence was determined with 100 nM E2. ( G ), Reciprocal coactivation of DAF-12 by sirtuins. SIRT1, Sir2 and Sir-2.1 were coexpressed in Hep3B with the DAF-12 reporter gene (lit-1k-TK-luc) alone or together with DAF-12. Cells were treated with DMSO or 1 µM each of either Δ 4 - or Δ 7 -dafachronic acid (DA). ( H ), DAF-12 coactivation by sirtuins is comparable with NcoA3/SRC-3 and PGC-1α. For all samples (A–H), β-gal expression was used as internal control. All luciferase data were normalized to β-gal activity levels. Datasets were plotted in duplicates and shown as means ± S.E.M; each graph is representative of at least three independent experiments. The first pair of columns on each graph shows the activity of the reporter gene alone treated with DMSO or ligand. Where indicated, coregulators (sirtuins, NcoA3/SRC-3, and PGC-1α) were cotransfected with reporter genes alone to determine intrinsic transcriptional activity. The statistical significance of differences in gene expression between DMSO and ligand-treated cells are * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001; where none of these is shown, differences are not significant.

Figure Legend Snippet: ( A ) SIRT1 elicits ER subtype-selectivity in liver cells, coactivating ERα but not ERβ; the NR-box is required for ligand-dependent coactivation. Wild-type SIRT1 or its NR-box mutant SIRT1ΔNRB and the STACs-AD expression vectors were cotransfected into Hep3B cells with VP16-ERα or VP16-ERβ and the ER reporter gene 3xERRE.EREluc. ( B ) Sirtuins are veritable ER coactivators. Hep3B cells were cotransfected with 3xERRE.EREluc and SIRT1, Sir2 and Sir-2.1, with or without ERα. ( C ) ER subtype selectivity by SIRT1 is similar to NcoA3/SRC-3. ( D ) SIRT1 and PGC-1α are independent veritable ER coactivators. ( E ) Comparision of ERα coactivation by the sirtuins NcoA3/SRC-3 and PGC-1α. As in ( D ) note the strong intrinsic transcriptional activity of PGC-1α compared with the sirtuins and NcoA3/SRC-3. ( F ) Sir-2.1 and Sir2 but not SIRT1 show relaxed specificity towards ERβ. In (A–F) Hep3B cells were cotransfected with sirtuins and the ER reporter (3xERRE.EREluc) alone and together with ERα or ERβ. Ligand-independent coactivation was determined by incubating cells with DMSO while ligand-dependence was determined with 100 nM E2. ( G ), Reciprocal coactivation of DAF-12 by sirtuins. SIRT1, Sir2 and Sir-2.1 were coexpressed in Hep3B with the DAF-12 reporter gene (lit-1k-TK-luc) alone or together with DAF-12. Cells were treated with DMSO or 1 µM each of either Δ 4 - or Δ 7 -dafachronic acid (DA). ( H ), DAF-12 coactivation by sirtuins is comparable with NcoA3/SRC-3 and PGC-1α. For all samples (A–H), β-gal expression was used as internal control. All luciferase data were normalized to β-gal activity levels. Datasets were plotted in duplicates and shown as means ± S.E.M; each graph is representative of at least three independent experiments. The first pair of columns on each graph shows the activity of the reporter gene alone treated with DMSO or ligand. Where indicated, coregulators (sirtuins, NcoA3/SRC-3, and PGC-1α) were cotransfected with reporter genes alone to determine intrinsic transcriptional activity. The statistical significance of differences in gene expression between DMSO and ligand-treated cells are * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001; where none of these is shown, differences are not significant.

Techniques Used: Mutagenesis, Expressing, Activity Assay, Luciferase

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Journal: Bioscience Reports

Article Title: A conserved mechanism of sirtuin signalling through steroid hormone receptors

doi: 10.1042/BSR20193535

Figure Lengend Snippet: ( A ) M2H assays were performed in HEK293 cells to determine interactions between VP16-ERα and SIRT1, Sir2 and Sir-2.1 tethered to Gal4DBD. ( B ) SIRT1-PGC-1α transcriptional complexes strongly interact with ERα and ERβ and overide Gal4 promoter repression by SIRT1 alone. M2H assay was repeated with Gal4DBD-SIRT1 and Gal4DBD-PGC-1α, and ERα or ERβ fused to VP16 activation domain. ( C ) M2H assay for interaction between the STACs-AD and VP16-ERα or VP16-ERβ. Gal4DBD-STACs-AD Wt or its NR-box mutant (ΔNRB) were cotransfected with the receptors as indicated. Schematic shows locations of Wt and mutant NR-boxes within the STACs-AD. Statistical significance of differences in interaction (B) or gene expression in response to DMSO or E2 (A–C) are shown with P values: * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001; differences are not significant where P values are not shown. 5x Gal4-luc and β-gal activities were determined as described and ER interaction with Gal4DBD was set as reference point. Data were plotted in duplicates as means ± S.E.M and are representative of three independent experiments.

Article Snippet: VP16-ER alpha (# 11351) and VP16-ER beta long isoform (#11352), p413TEF-Sir2 (# 51742), 3xERRE/ERE-luciferase (# 37852), SIRT1.1 (# 13735), Flag-SIRT1 (# 1791), Flag-SIRT1 H 363 Y (# 1792) and Gal4-PGC-1 alpha (# 8892) were purchased from Addgene (Cambridge, MA, U.S.A.). pET28-Sir-2.1 was kindly provided by Leonard Guarente (MIT, Cambridge, MA, U.S.A.) while VP16-DAF-12 and its reporter gene lit -1K-TK-luc [ ] were kindly offered by David Mangelsdorf (UT Southwestern Medical Centre, Dallas, TX, U.S.A.).

Techniques: Activation Assay, Mutagenesis, Expressing

Journal: Bioscience Reports

Article Title: A conserved mechanism of sirtuin signalling through steroid hormone receptors

doi: 10.1042/BSR20193535

Figure Lengend Snippet: Increasing doses (50, 100 and 200 ng) of Gal4DBD fusion peptides of wild-type (NRBWt) and mutant (NRBMt) SIRT1 NR-boxes were tested in M2H assays in HEK293 cells for interaction with ( A ) VP16-ERα and ( B ) VP16-ERβ; the reporter gene was 5x Gal4-luc. Luciferase expression was normalized to β-gal activity. Graphs were plotted by setting the activity of Gal4DBD interaction with the ERs as reference. P values show the significance of differences in ER interaction, comparing NRBWt with NRBMt; only P values ≤ 0.05 are significant. ( C–F ) Modified M2H assay of SIRT1 NR-box peptide binding specificity by competitive inhibition with wild-type (C,E) and mutant (D,F) NR-box peptides. Interactions were determined through dose-dependent repression of ERα (C,D) and ERβ (E,F) signalling; antagonism was observed with wild-type (C,E) but not with mutant (D,F) NR-box peptides. For panels (C–F), MCF-7 cells were cotransfected with VP16-ERα or VP16-ERβ, the ER reporter gene instead of 5x Gal4-luc, and increasing amounts (50, 100 and 200 ng) of Gal4DBD SIRT1 NR-box peptides. Cells were treated with DMSO or 100 nM E2 and ER reporter gene expression was normalized to β-gal internal control. Fold activation was determined from the activity of Gal4DBD (negative control) set at a value of 1. P values show significant ( P ≤ 0.05) differences between DMSO and E2-treated cells. Data are representative of three independent assays.

Techniques: Mutagenesis, Luciferase, Expressing, Activity Assay, Modification, Binding Assay, Inhibition, Activation Assay, Negative Control

Journal: Bioscience Reports

Article Title: A conserved mechanism of sirtuin signalling through steroid hormone receptors

doi: 10.1042/BSR20193535

Figure Lengend Snippet: ( A ) SIRT1 elicits ER subtype-selectivity in liver cells, coactivating ERα but not ERβ; the NR-box is required for ligand-dependent coactivation. Wild-type SIRT1 or its NR-box mutant SIRT1ΔNRB and the STACs-AD expression vectors were cotransfected into Hep3B cells with VP16-ERα or VP16-ERβ and the ER reporter gene 3xERRE.EREluc. ( B ) Sirtuins are veritable ER coactivators. Hep3B cells were cotransfected with 3xERRE.EREluc and SIRT1, Sir2 and Sir-2.1, with or without ERα. ( C ) ER subtype selectivity by SIRT1 is similar to NcoA3/SRC-3. ( D ) SIRT1 and PGC-1α are independent veritable ER coactivators. ( E ) Comparision of ERα coactivation by the sirtuins NcoA3/SRC-3 and PGC-1α. As in ( D ) note the strong intrinsic transcriptional activity of PGC-1α compared with the sirtuins and NcoA3/SRC-3. ( F ) Sir-2.1 and Sir2 but not SIRT1 show relaxed specificity towards ERβ. In (A–F) Hep3B cells were cotransfected with sirtuins and the ER reporter (3xERRE.EREluc) alone and together with ERα or ERβ. Ligand-independent coactivation was determined by incubating cells with DMSO while ligand-dependence was determined with 100 nM E2. ( G ), Reciprocal coactivation of DAF-12 by sirtuins. SIRT1, Sir2 and Sir-2.1 were coexpressed in Hep3B with the DAF-12 reporter gene (lit-1k-TK-luc) alone or together with DAF-12. Cells were treated with DMSO or 1 µM each of either Δ 4 - or Δ 7 -dafachronic acid (DA). ( H ), DAF-12 coactivation by sirtuins is comparable with NcoA3/SRC-3 and PGC-1α. For all samples (A–H), β-gal expression was used as internal control. All luciferase data were normalized to β-gal activity levels. Datasets were plotted in duplicates and shown as means ± S.E.M; each graph is representative of at least three independent experiments. The first pair of columns on each graph shows the activity of the reporter gene alone treated with DMSO or ligand. Where indicated, coregulators (sirtuins, NcoA3/SRC-3, and PGC-1α) were cotransfected with reporter genes alone to determine intrinsic transcriptional activity. The statistical significance of differences in gene expression between DMSO and ligand-treated cells are * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001; where none of these is shown, differences are not significant.

Techniques: Mutagenesis, Expressing, Activity Assay, Luciferase

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