Journal:

Article Title: Phosphorylation of Ser 158 regulates inflammatory redox-dependent hepatocyte nuclear factor-4? transcriptional activity

doi: 10.1042/BJ20051730

Figure Lengend Snippet: HNF4α serine/threonine phosphorylation sites Phosphopeptide analysis of putative serine and threonine phosphorylation sites in HNF4α under various treatment conditions. The bold underlined amino acids represent identified phosphorylation targets.

Article Snippet: Cells (1×10 6 ) were transfected with 10 μg of WT-HNF4α expression vector, and/or mutant HNF4α vectors using Lipofectin® (Invitrogen) according to the manufacturer's recommendation.

Techniques:

Journal:

Article Title: Phosphorylation of Ser 158 regulates inflammatory redox-dependent hepatocyte nuclear factor-4? transcriptional activity

doi: 10.1042/BJ20051730

Figure Lengend Snippet: (A) ChIP assay of iNOS promoter HNF4α binding. Chromatin from hepatocytes was fixed and immunoprecipitated using the ChIP assay kit as recommended by the manufacturer. Goat anti-human polyclonal HNF4α antibody (5 μg, Santa Cruz Biotechnology) was used for each immunoprecipitation; 5 μl of rabbit IgG served as a control. The DNA was recovered and subjected to analysis by PCR. The primers had the following sequence: 5′-tgaccaattgactggtatgtgtg-3′ (sense strand) and 5′-gctgggctggggagatggctga-3′ (antisense strand) to yield a PCR product of 280 bp. The input fraction corresponded to 0.1% of the chromatin solution before immunoprecipitation. After DNA purification, the presence of the selected DNA sequence was assessed by PCR. The blot is representative of four experiments. H2O2 (50 μM); STA, staurosporine (0.5 μM). (B) EMSA analysis of in vitro WT and mutant HNF4α binding. EMSAs were performed using 10 μg of FLAG-tagged WT or mutant HNF4 peptide. Unlabelled target oligonucleotide was added at 200 M excess in specific or non-specific competition assays (Spec Comp or Nonspec Comp respectively). For supershift experiments, FLAG antibody was included in the binding reaction. Probe was prepared by end-labelling the WT 28 bp double-stranded ARE (antioxidant response element) with [γ-32P]ATP (2500 Ci/mmol) using T4 polynucleotide kinase, followed by gel purification on 15% polyacrylamide. The gel is representative of five experiments. Ab, antibody.

Article Snippet: Cells (1×10 6 ) were transfected with 10 μg of WT-HNF4α expression vector, and/or mutant HNF4α vectors using Lipofectin® (Invitrogen) according to the manufacturer's recommendation.

Techniques: Binding Assay, Immunoprecipitation, Sequencing, DNA Purification, In Vitro, Mutagenesis, Gel Purification

Journal:

Article Title: Phosphorylation of Ser 158 regulates inflammatory redox-dependent hepatocyte nuclear factor-4? transcriptional activity

doi: 10.1042/BJ20051730

Figure Lengend Snippet: Cells were transfected using the Lipofectamine™ technique. After cells were washed twice with MEM, 10 μg of plasmid DNA containing the iNOS promoter construct (1845 bp; GenBank® accession no. X95629) coupled with a CAT reporter gene were added. In selected instances, an empty vector or an HNF4α expression vector (10 μg) encoding FLAG-tagged WT or mutant variants was co-transfected with the iNOS promoter plasmid construct. After 24 h, cells were stimulated with IL-1β (1000 units/ml) in the presence and absence of H2O2 (50 μM). Unstimulated cells served as controls. After 6 h, the supernatant was assayed for CAT activity using a CAT ELISA technique. Transfection efficiency was normalized by co-transfection of a β-galactosidase reporter gene with a constitutively active early SV40 promoter. All values are expressed as pg of CAT/mg of protein. Results are expressed as means±S.E.M. for three experiments (*P<0.01 versus control, IL-1β- or H2O2-treated cells).

Article Snippet: Cells (1×10 6 ) were transfected with 10 μg of WT-HNF4α expression vector, and/or mutant HNF4α vectors using Lipofectin® (Invitrogen) according to the manufacturer's recommendation.

Techniques: Transfection, Plasmid Preparation, Construct, Expressing, Mutagenesis, Activity Assay, Enzyme-linked Immunosorbent Assay, Cotransfection

Journal:

Article Title: Phosphorylation of Ser 158 regulates inflammatory redox-dependent hepatocyte nuclear factor-4? transcriptional activity

doi: 10.1042/BJ20051730

Figure Lengend Snippet: COS-1 cells were transfected with empty vector or FLAG-tagged WT or mutant (S158A or S304A) HNF4α expression vectors. Cells were treated with IL-1β+H2O2 in the presence or absence of increasing concentrations of p38, PKA or PKC inhibitors. Unstimulated cells served as a control. EMSAs were performed using 10 μg of isolated HNF4 peptide. For the supershift experiments, anti-FLAG antibody (Kodak) was included in the binding reaction. Probe was prepared by end-labelling WT 28 bp double-stranded ARE with [γ-32P]ATP (2500 Ci/mmol) using T4 polynucleotide kinase, followed by gel purification on 15% polyacrylamide. The gel is representative of five experiments. NP, not performed; Ab, antibody.

Article Snippet: Cells (1×10 6 ) were transfected with 10 μg of WT-HNF4α expression vector, and/or mutant HNF4α vectors using Lipofectin® (Invitrogen) according to the manufacturer's recommendation.

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Expressing, Isolation, Binding Assay, Gel Purification

Journal:

Article Title: Phosphorylation of Ser 158 regulates inflammatory redox-dependent hepatocyte nuclear factor-4? transcriptional activity

doi: 10.1042/BJ20051730

Figure Lengend Snippet: COS-1 cells were transfected with empty vector or FLAG-tagged WT or mutant (S158A or S158D) HNF4α expression vectors. Cells were treated as indicated. Unstimulated cells served as a control. EMSAs were performed using 10 μg of isolated HNF4 peptide. Probe was prepared by end-labelling the WT 28 bp double-stranded ARE with [γ-32P]ATP (2500 Ci/mmol) using T4 polynucleotide kinase, followed by gel purification on 15% polyacrylamide. The gel is representative of three experiments.

Article Snippet: Cells (1×10 6 ) were transfected with 10 μg of WT-HNF4α expression vector, and/or mutant HNF4α vectors using Lipofectin® (Invitrogen) according to the manufacturer's recommendation.

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Expressing, Isolation, Gel Purification

Journal:

Article Title: Phosphorylation of Ser 158 regulates inflammatory redox-dependent hepatocyte nuclear factor-4? transcriptional activity

doi: 10.1042/BJ20051730

Figure Lengend Snippet: (A) In vitro phosphorylation of HNF4α. Purified FLAG-tagged WT or mutant HNF4α protein was incubated with 1 μl of protein kinase (p38, JNK, or ERK1/2). The reaction products were immunoprecipitated with anti-FLAG antibody at 0 and 20 min, followed by separation on SDS/4–20% polyacrylamide gels; the gel was then dried and analysed by autoradiography. The blot is representative of three experiments. (B) Transient transfection analysis of iNOS promoter activity in COS-1 cells. Cells were transfected using the Lipofectamine™ technique. After cells were washed twice with medium, 10 μg of plasmid DNA containing the iNOS promoter construct (1845 bp; GenBank® accession no. X95629) coupled with a CAT reporter gene were added. In selected instances, an empty vector or an HNF4α expression vector (10 μg) encoding FLAG-tagged WT or mutant variants was co-transfected with the iNOS promoter plasmid construct. In selected instances, the p38 kinase inhibitor was also added. Cells were stimulated with IL-1β (1000 units/ml) in the presence and absence of H2O2 (50 μM). Unstimulated cells served as controls. After 6 h, the supernatant was assayed for CAT activity using a CAT ELISA technique. Transfection efficiency was normalized by co-transfection of a β-galactosidase reporter gene with a constitutively active early SV40 promoter. All values are expressed as pg of CAT/mg of protein. Results are expressed as means±S.E.M. for three experiments (*P<0.01 versus control or H2O2-treated cells; **P<0.01 versus control, IL-1β- or H2O2-treated cells).

Article Snippet: Cells (1×10 6 ) were transfected with 10 μg of WT-HNF4α expression vector, and/or mutant HNF4α vectors using Lipofectin® (Invitrogen) according to the manufacturer's recommendation.

Techniques: In Vitro, Purification, Mutagenesis, Incubation, Immunoprecipitation, Autoradiography, Transfection, Activity Assay, Plasmid Preparation, Construct, Expressing, Enzyme-linked Immunosorbent Assay, Cotransfection

Journal:

Article Title: Phosphorylation of Ser 158 regulates inflammatory redox-dependent hepatocyte nuclear factor-4? transcriptional activity

doi: 10.1042/BJ20051730

Figure Lengend Snippet: HepG2 cells were transfected with FLAG-tagged WT-HNF4α or S158A-HNF4α by Lipofectin® (Invitrogen) and exposed to IL-1β+H2O2. Immunoprecipitation (IP) was performed with anti-FLAG M2–agarose affinity gel (Sigma). Polyclonal anti-PC4 antibody (Santa Cruz Biotechnology) was used for immunoblot (IB) analysis. Membranes were then exposed to secondary antibody coupled with horseradish peroxidase and visualized. The blot is representative of three experiments.

Article Snippet: Cells (1×10 6 ) were transfected with 10 μg of WT-HNF4α expression vector, and/or mutant HNF4α vectors using Lipofectin® (Invitrogen) according to the manufacturer's recommendation.

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: PPAR Research

Article Title: The Peroxisomal 3-keto-acyl-CoA thiolase B Gene Expression Is under the Dual Control of PPAR α and HNF4 α in the Liver

doi: 10.1155/2010/352957

Figure Lengend Snippet: HNF4 α is a master gene for the hepatic expression of Thb . (a) Expression of hepatic Thb in HNF4 α null ( Hnf4 loxP/loxP Alfp.cre ; loxP/loxP) and control ( Hnf4 loxP/+ Alfp.cre ; loxP/+) mouse embryos was determined by semiquantitative RT-PCR analysis ( n = 3 per group). Hprt mRNA livers were evaluated and used as internal standard of loading. Tha : thiolase A; Thb : thiolase B; Hnf4α : Hepatocyte Nuclear Factor-4 alpha; Hprt : Hypoxanthine-guanine PhosphoRibosyl Transferase. (b) Mouse Hepa 1.6 hepatoma cells were transfected with expression vectors for WT HNF4 α or dominant negative form of HNF4 α (DN HNF4). Expression of Thb , Tha , and apolipoprotein AII ( ApoA-II ) was determined by quantitative RT-PCR. Crude results were standardized against 36B4 mRNA levels. Levels of gene expression in Hepa 1.6 cells transfected with empty pcDNA3.1 vector serve as reference point and are given the arbitrary value of 1.0. Values are mean of three independent experiments ±SEM. Apolipoprotein AII (ApoAII) gene expression was used as a positive control of experiment. Significantly different compared to control (transfection with empty pcDNA3.1 vector) with * P < .05 and ** P < .01 by one-way ANOVA test. n.s.: no statistically different compared to control.

Article Snippet: The HNF4 α (sc-6556), PPAR α (sc-9000x), RXR α (sc-774), as well as the secondary (donkey anti-goat, sc-2020) antibodies used were all purchased from Santa-Cruz Biotechnology Inc.

Techniques: Expressing, Control, Reverse Transcription Polymerase Chain Reaction, Transfection, Dominant Negative Mutation, Quantitative RT-PCR, Gene Expression, Plasmid Preparation, Positive Control

Journal: PPAR Research

Article Title: The Peroxisomal 3-keto-acyl-CoA thiolase B Gene Expression Is under the Dual Control of PPAR α and HNF4 α in the Liver

doi: 10.1155/2010/352957

Figure Lengend Snippet: TB PPRE3 is a novel binding site for the nuclear receptor HNF4 α. (a) Binding of HNF4 α to native radiolabelled ( 32 P) TB PPRE3 was determined by gel shift assay. A double strand oligonucleotide containing ( 32 P) TB PPRE3 was incubated with lysates of transfected (by the expression vector HNF4 α ) COS-7 cells. Fold excess of specific (Sp.) cold probe (PPRE of the peroxisomal ACOX-I gene) was used for data shown lanes 3, 4 and 5. Nonspecific (Non Sp.) cold probe (Sp1) was used for data shown lanes 6, 7 and 8. Binding complexes were resolved on a 6% non-denaturing polyacrylamide gel. The arrow indicates the specific binding of HNF4 α . The star indicates nonspecific binding. (b) Supershift assay (lane 2) was performed with an antibody directed against HNF4 α . (c) COS-7 cells were transfected with increasing amounts (0 to 250 ng) of expression vectors encoding WT HNF4 α with a Luc reporter vector containing TB PPRE3. (d) COS-7 cells were transfected with increasing amounts (0 to 250 ng) of expression vectors encoding wild-type HNF4 α with a Luc reporter vector containing one copy of the DR1 localized in intron 5 of the mouse version of Thb (+4083 +4095 bp). Values are mean of three independent experiments ±SEM. Significantly different compared to control (transfection with empty pcDNA3.1 vector) with * P < .05 by one-way ANOVA test.

Article Snippet: The HNF4 α (sc-6556), PPAR α (sc-9000x), RXR α (sc-774), as well as the secondary (donkey anti-goat, sc-2020) antibodies used were all purchased from Santa-Cruz Biotechnology Inc.

Techniques: Binding Assay, Gel Shift, Incubation, Transfection, Expressing, Plasmid Preparation, Control

Journal: PPAR Research

Article Title: The Peroxisomal 3-keto-acyl-CoA thiolase B Gene Expression Is under the Dual Control of PPAR α and HNF4 α in the Liver

doi: 10.1155/2010/352957

Figure Lengend Snippet: PPAR α transactivation from TB PPRE3 is markedly enhanced by HNF4 α . (a) Transactivation assays were performed in COS-7 and HeLa cells (b) or in Hepa 1.6 and H4IIEC3 cells with a Luc reporter vector containing isolated rTB PPRE3. These constructs were transfected together with expression vectors for both mouse PPAR α (pSG5-mPPAR α ) and RXR α (pSG5-mRXR α ) in absence (white bars) or presence (black bars) of Wy (10 μ M). Cotransfection with pcDNA3.1 WT hHNF4 α was performed as indicated. Note that the minimal promoter of the thiolase B gene was used instead of the globin gene promoter which was inactive in H4IIEC3 cells. (c) Transactivation assays were performed in COS-7 or Hepa 1.6 cells with a Luc reporter vector containing isolated ACOX-I PPRE. Normalized luciferase activity of each construct in the absence of PPAR α and ligand was set at 1. Values are mean of four independent experiments ±SEM. DMSO was used as vehicle. Significantly different compared to control (transfection with a combination of empty pcDNA3.1 and pSG5 vectors) with ** P < .01 and *** P < .001 by one-way ANOVA test. Significantly different between PPAR α /RXR α and PPAR α /RXR α + HNF4 α with $$ P < .01 and $$$ P < .001 by one-way ANOVA test.

Article Snippet: The HNF4 α (sc-6556), PPAR α (sc-9000x), RXR α (sc-774), as well as the secondary (donkey anti-goat, sc-2020) antibodies used were all purchased from Santa-Cruz Biotechnology Inc.

Techniques: Plasmid Preparation, Isolation, Construct, Transfection, Expressing, Cotransfection, Luciferase, Activity Assay, Control

Journal: PPAR Research

Article Title: The Peroxisomal 3-keto-acyl-CoA thiolase B Gene Expression Is under the Dual Control of PPAR α and HNF4 α in the Liver

doi: 10.1155/2010/352957

Figure Lengend Snippet: Binding of HNF4 α to TB PPRE3 is dispensable for the cooperation with PPAR α /RXR α . (a) Positions of mutations used in this study. A scheme of HNF4 α structure with the various domains is given: DBD, DNA-binding domain; LBD, ligand-binding domain; AF2, activation function 2 module. (b) Transactivation assays were performed in COS-7 cells with a Luc reporter vector containing isolated TB PPRE3 (b) or mouse ACOX-I PPRE (c). These constructs were cotransfected with expression vectors for both mouse PPAR α (pSG5-mPPAR α ) and RXR α (pSG5-mRXR α ) together with an expression vector encoding either wild-type HNF4 α (HNF4 α 2 WT), a first (D126Y HNF4 α 2) or a second deficient form (DN HNF4) of HNF4 α for DNA binding in absence (white bars) or presence (black bars) of Wy (10 μ M). Values are mean of three independent experiments ±SEM. DMSO was used as vehicle. Significantly different between PPAR α /RXR α and PPAR α /RXR α + HNF4 α with ** P < .01 and *** P < .001 by one-way ANOVA test.

Article Snippet: The HNF4 α (sc-6556), PPAR α (sc-9000x), RXR α (sc-774), as well as the secondary (donkey anti-goat, sc-2020) antibodies used were all purchased from Santa-Cruz Biotechnology Inc.

Techniques: Binding Assay, Ligand Binding Assay, Activation Assay, Plasmid Preparation, Isolation, Construct, Expressing

Journal: PPAR Research

Article Title: The Peroxisomal 3-keto-acyl-CoA thiolase B Gene Expression Is under the Dual Control of PPAR α and HNF4 α in the Liver

doi: 10.1155/2010/352957

Figure Lengend Snippet: Binding of PPAR α /RXR α to TB PPRE3 is not enhanced by HNF4 α . (a) Schematic outline of the DAPA procedure. (b) DAPA was performed with nuclear extracts of transfected COS-7 cells (with either HNF4 α , PPAR α , or RXR α ) using the biotinylated TB PPRE3 oligonucleotides. Complexed proteins were resolved by SDS-PAGE and revealed by Western blot using an anti-PPAR α or anti-HNF4 α antibody . (d) Coimmunoprecipitation of RXR α and HNF4 α . Nuclear extracts from transfected COS-7 cells were immunoprecipitated (IP) with PPAR α or RXR α antibodies, as indicated. The total plasmid amount was adjusted with pCDNA3.1 parent vector to 8 μ g for each 100-mm transfection with Exgen 500. Cells were incubated in DMEM 10% FCS with 10 μ M Wy for 48 h. The presence of HNF4 α protein in the immunopurified material (100 μ g of nuclear protein) was detected by Western blot assay using anti-HNF4 α antibody. NE: Nuclear Extracts. Lane 1: Only 4 μ g of nuclear protein were loaded.

Article Snippet: The HNF4 α (sc-6556), PPAR α (sc-9000x), RXR α (sc-774), as well as the secondary (donkey anti-goat, sc-2020) antibodies used were all purchased from Santa-Cruz Biotechnology Inc.

Techniques: Binding Assay, Transfection, SDS Page, Western Blot, Immunoprecipitation, Plasmid Preparation, Incubation

Journal: PPAR Research

Article Title: The Peroxisomal 3-keto-acyl-CoA thiolase B Gene Expression Is under the Dual Control of PPAR α and HNF4 α in the Liver

doi: 10.1155/2010/352957

Figure Lengend Snippet: The PPAR α -regulated induction of Thb is potentiated by HNF4 α in the liver. Hepatic mRNA levels in the liver-specifically HNF4 α -disrupted (HNF4 α ∆L) and HNF4 α F/F-fed control or Wy14,643-containing diet (0.1% w/w) for five days. Total RNA extracted from livers of these mice were subjected to real-time PCR analysis. The expression signals from the WT mice that did not receive Wy were arbitrarily set at 1. The results are shown as a relative expression to β -actin mRNA levels as normalization control. Error bars represent standard error (SE) and data are expressed as the mean ± S.E. ( n = 4 for each condition). Significant effects were calculated using two-way ANOVA test for the genotype (G), Wy14,643 (Wy) and the interaction between both parameters (I). Results are indicated at the top of each figure. In bold, parameters that are under the cutoff for statistical significance ( P value of .05 or below). Wy: Wy14,643.

Article Snippet: The HNF4 α (sc-6556), PPAR α (sc-9000x), RXR α (sc-774), as well as the secondary (donkey anti-goat, sc-2020) antibodies used were all purchased from Santa-Cruz Biotechnology Inc.

Techniques: Control, Real-time Polymerase Chain Reaction, Expressing

Journal:

Article Title: Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4? dimerisation and transcriptional activity

doi: 10.1093/nar/gkg850

Figure Lengend Snippet: Structure-based amino acid sequence alignment of H7 of nuclear receptor LBDs (adapted from references 27–29). Usual names of nuclear receptors are indicated on the left side whereas names proposed by the Nuclear Receptor Nomenclature Committee are indicated on the right side. The arrow and boxed R indicate the glutamate and arginyl residues specifically found in members of the NR2 subfamily. Positions of the functional domains, of the I-box and of activation function 2 (AF-2) are shown. *, position in isoform 2 of HNF4α (HNF4α2), which was used in this study.

Article Snippet: Wild-type and mutated HNF4α were in vitro synthesised in reticulocyte lysates (Promega).

Techniques: Sequencing, Functional Assay, Activation Assay

Journal:

Article Title: Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4? dimerisation and transcriptional activity

doi: 10.1093/nar/gkg850

Figure Lengend Snippet: E262 and R258 are involved in HNF4α2 dimerisation in solution. (A and B) Analyses by co-immunoprecipitation assays and GST pull-down assays, respectively, of dimerisation between immobilised wild-type HNF4α2 fused to c-myc or GST (c-myc-HNF4α2 WT or GST-HNF4α2 WT) and wild-type or mutated [35S]methionine-labelled HNF4α2. Graphs in (A) and (B) indicate means ± SE of HNF4α2 mutant binding relative to that of the wild-type protein from three independent experiments. Inputs were taken into account for binding quantifications. (C) Dimerisation, analysed by co-immunoprecipitation assays of HNF4α2 WT, -ΔE262 or -E262A. For each assay, [35S]methionine-labelled HNF4α2 was incubated with the same protein fused to the c-myc tag. Control of synthesis of c-myc-HNF4α2 WT and mutated proteins is shown in the insert. (D) Dimerisation, analysed by GST pull-down assays of HNF4α2 WT, -ΔE262 and -E262A. For each assay, [35S]methionine-labelled HNF4α2 was incubated with the same protein fused to GST. Pull-down assays were performed in the indicated ionic strength conditions. The graph indicates means ± SE of HNF4α binding at 300 or 600 mM KCl relative to binding at 100 mM KCl (set to 100%) from three independent experiments. (E) Dimerisation, analysed by co-immunoprecipitation assays, between immobilised c-myc-HNF4α2 WT and [35S]methionine-labelled HNF4α2 WT or -R258M. The graph indicates mean ± SE of HNF4α2-R258M binding relative to that of the wild-type protein from four independent experiments. Inputs were taken into account for binding quantifications.

Article Snippet: Wild-type and mutated HNF4α were in vitro synthesised in reticulocyte lysates (Promega).

Techniques: Immunoprecipitation, Mutagenesis, Binding Assay, Incubation

Journal:

Article Title: Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4? dimerisation and transcriptional activity

doi: 10.1093/nar/gkg850

Figure Lengend Snippet: Mutations of E262 and R258 residues do not impair HNF4α2 DNA binding. (A) DNA binding of HNF4α2 mutants to the 32P-labelled HNF4α response element of the HNF1α promoter (HNF1 site). Control of in vitro synthesis of wild-type and mutated HNF4α2, used in EMSA, is shown in the insert (values on the right end indicate molecular size markers). The graph indicates means ± SE of mutated HNF4α2 DNA binding relative to that of the wild-type protein from three independent experiments. (B) Specificity of binding. Unprogrammed reticulocyte lysate (mock) yielded no shifted band. Supershifting was performed in the presence of the specific α455 HNF4α antiserum. (C) EMSA performed with a constant amount of HNF4α2 WT or HNF4α2-E262A and increasing amounts of labelled HNF1 probe. (D) HNF4α2-E262A did not bind as a monomer to the half-site of the HNF4α response element (HNF1 mt). (E) Competition experiments with COUP-TFII ΔAB. EMSA were performed on the HNF4 response element of the apoCIII promoter (CIIIB site) using in vitro synthesised HNF4α2 WT or HNF4α2-E262A and increasing amounts of the competitor COUP-TFII ΔAB. The amount of reticulocyte lysate in each lane was held constant by the appropriate addition of unprogrammed lysate. The positions of HNF4α2 and COUP-TFII ΔAB homodimers bound to DNA are indicated.

Article Snippet: Wild-type and mutated HNF4α were in vitro synthesised in reticulocyte lysates (Promega).

Techniques: Binding Assay, In Vitro

Journal:

Article Title: Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4? dimerisation and transcriptional activity

doi: 10.1093/nar/gkg850

Figure Lengend Snippet: Differential effects on dimerisation in solution and DNA binding of mutations in H7 and H10 of the HNF4α LBD. (A) Dimerisation, analysed by co-immunoprecipitation assays, between immobilised c-myc-HNF4α2 WT and [35S]methionine-labelled HNF4α2 WT or -E327M. The graph indicates mean ± SE of HNF4α2-E327M binding relative to that of the wild-type protein from three independent experiments. Inputs were taken into account for binding quantifications. (B) Dimerisation, analysed by GST pull-down assays, between immobilised GST-HNF4α2 WT and [35S]methionine-labelled HNF4α2 WT, -R258M and -E327M. Pull-down assays were performed in various ionic strength conditions as in Figure ​Figure2D.2D. The graph indicates means ± SE of mutant binding relative to that of the wild-type HNF4α from three independent experiments. Inputs were taken into account for binding quantifications. (C) Dimerisation, analysed by co-immunoprecipitation assays using the α455 antiserum, between immobilised HNF4α2 WT, -R258M, -E262A or -E327M and [35S]methionine-labelled HNF4α3, which is not recognised by the α455 antiserum. Control of HNF4α2 protein synthesis is shown in the insert. The graph indicates means ± SE of HNF4α3 retention by HNF4α2 mutants relative to HNF4α3 retention by HNF4α2 WT from four independent experiments. Control of protein synthesis was taken into account for binding quantifications. (D) DNA binding of HNF4α2-E327M on the HNF1 site, analysed by EMSA performed as in Figure ​Figure3A.3A. Supershifts were obtained in the presence of the α455 HNF4α antiserum as indicated. Control of protein synthesis is shown in the insert. The graph indicates mean ± SE of HNF4α2-E327M DNA binding relative to that of the wild-type protein from three independent experiments.

Article Snippet: Wild-type and mutated HNF4α were in vitro synthesised in reticulocyte lysates (Promega).

Techniques: Binding Assay, Immunoprecipitation, Mutagenesis

Journal:

Article Title: Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4? dimerisation and transcriptional activity

doi: 10.1093/nar/gkg850

Figure Lengend Snippet: Deletion of E262 strongly affects HNF4α2 transcriptional activity. HeLa (A), HEK 293 (B) and COS-1 cells (C) were transiently transfected with 12.5 ng of expression vector for wild-type or mutated HNF4α2 or the corresponding empty vector (–) together with 250 ng of HNF1α promoter construct. Fold induction refers to the activity with no HNF4α2 derivative (–), which was set to 1. Results are means ± SE of three independent experiments performed in triplicate. **, P = 0.0015, 0.0060 and 0.0018 for the ΔE262 mutant in (A–C), respectively; ***, P < 0.0001 for the ΔD261 mutant in (A). (D) Western blotting of HeLa cell extracts. (E) HNF4α-ΔE262 does not exhibit a dominant-negative activity on wild-type HNF4α. COS-1 cells were transfected as in (C), except that equal amounts of wild-type HNF4α and HNF4α-ΔE262 or control vector (–) were co-transfected. (F and G) Effects of substitution mutations on the dominant-negative activity of HNF4α-ΔAF-2. COS-1 cells were transfected as in (C), except that in (F) plasmids expressing wild-type, E262A or E262K HNF4α were co-transfected with an equal amount of vector expressing HNF4α-ΔAF-2 or the control vector (–), whereas in (G) pcDNA3 HNF4α2 WT was co-transfected with an equal amount of vectors expressing HNF4α-ΔAF-2 or HNF4α-ΔAF-2-E262A or the control vector (–). Activation of the HNF1α promoter is expressed relative to that obtained when only full-length proteins were expressed. Results are means ± SE of three independent experiments performed in triplicate. **, P = 0.0040 in (F); ***, P < 0.0001 in (F); *, P = 0.0278 in (G).

Article Snippet: Wild-type and mutated HNF4α were in vitro synthesised in reticulocyte lysates (Promega).

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Construct, Mutagenesis, Western Blot, Dominant Negative Mutation, Activation Assay

Journal:

Article Title: Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4? dimerisation and transcriptional activity

doi: 10.1093/nar/gkg850

Figure Lengend Snippet: Deletion of E262 markedly decreases recruitment of transcriptional partners. (A) GST pull-down assays were performed using GST-SRC-1a (570–780), GST-p300 (340–528), GST-PGC-1 (36–797) or GST-COUP-TFII and [35S]methionine-labelled WT or mutated HNF4α2. Inputs correspond to 5 or 2% (for the experiment with GST-SRC-1a) of amounts of labelled proteins used in the assays. (B and C) Effects of mutations of the E262 residue on the enhancement of HNF4α2 transcriptional activity by p300 and COUP-TFII, respectively. HeLa cells were transiently transfected with 12.5 ng of wild-type or mutated HNF4α2 expression vector, 250 ng of HNF1α promoter construct and 250 ng of empty control vector (white bars) or expression vectors (black bars) for p300 or COUP-TFII. Shown are per cent enhancements of wild-type and mutated HNF4α activities. Results are means ± SE of three independent experiments performed in triplicate. *, P = 0.0109; **, P = 0.0037.

Article Snippet: Wild-type and mutated HNF4α were in vitro synthesised in reticulocyte lysates (Promega).

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