32 research outputs found

    In silico analysis of mouse PXR proximal promoter.

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    <p>The nucleotide sequence of the 5′ flanking genomic DNA of the mouse PXR gene is shown along with consensus sequences for potential DNA-protein binding sites. Transcription initiation site, +1, is denoted by an arrow. PU.1, purine rich box binding element; Ets-1, Ets binding element; GR, glucocorticoid receptor binding element; ER, estrogen receptor binding element; VDR, vitamin D receptor binding element; COUP-TF, chicken ovalbumin upstream promoter binding element; NF-AT, nuclear factor of activated T cells binding element; AP-1, activator protein binding element; HNF1/3/4, hepatocyte nuclear factor binding element; AR, androgen receptor binding element; RXR/RAR, retinoid X/acid receptor binding site; T3R, thyroid hormone receptor binding element; PPAR, peroxisome proliferator-activated receptor binding element; GATA, gata binding element; LEF, lymphoid enhancer factor binding element; LyF, lymphoid factor binding element; c-Myb, myb (myeloblast) binding element; NF-1, nuclear factor binding element. Region marked with red color shows putative binding sites for different transcription factors and this region is further used for EMSA analysis.</p

    Binding of β-catenin/LEF transcription factors to −243/−219 mouse PXR promoter region. A

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    <p>) Radiolabeled −243/−219 oligonucleotide was incubated with 15 μg of Hepa 1–6 nuclear lysate (lane 2). For competition experiments, unlabelled −243/−219 mouse PXR (lanes 3 and 4), unlabelled −297/−163 mouse PXR (lanes 5 and 6) and unlabelled non-self oligonucleotides (lanes 7 and 8) were added to the reactions in 50- and 200-fold molar excess. <b>B</b>) Antibodies against β-catenin (lane 2), LEF-1(lane 3) or pre-immune IgG (lane 1) were added to the DNA-protein complexes and incubated for an additional 15 minutes at room temperature. Supershifted bands are shown with an open arrow. NE = nuclear extract.</p

    EMSA of mouse PXR proximal promoter.

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    <p>An end-labeled 134 bp promoter fragment (−297/−163) was incubated with AML-12 whole cell lysate. Lane <b>1</b>: free probe; Lanes <b>2, 3</b> & <b>4</b>: binding reactions performed with increasing amount of 3 μg, 6 μg and 12 μg of AML-12 lysate respectively. DNA-protein complexes are shown with arrows.</p

    Regulation of mouse PXR proximal promoter by members of different families of transcription factors. A

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    <p>) The cultured Hepa1-6 cells were co-transfected with p-543/+54-Luc construct and various other expression plasmids encoding for different transcription factors, as indicated in the graph. Following expression period of 24 hours, cell lysates were prepared from the transfected Hepa 1–6 cells and luciferase and β-galactosidase activities were determined. <b>B</b>) Hepa 1–6 cells were transiently co-transfected with the p-543/+43-Luc construct together with WT-Ets-1 or DN-Ets-1 expression plasmids. Luciferase values were normalized for transfection efficiency with β-galactosidase values and are expressed as relative fold change with respect to pGL3 basic promoter-less vector. Data represent the mean ± SE of three different experiments. Asterisks (*) signify luciferase values that differed significantly from the pcDNA transfected cells (P<0.05 in Student's T-test).</p

    Sequence specific binding of Ets and LEF transcription factors to −297/−163 mouse proximal PXR promoter. A

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    <p>) A 134 bp (−297 to −163) PCR amplified fragment was radiolabeled and incubated with Hepa 1–6 cell lysate and competition assays were performed with 50- to 100-fold molar excess of unlabelled self-nucleotides (lanes 3 and 4), unlabelled oligonucleotide −283/−252 (lanes 5 and 6) and unlabelled oligonucleotide −243/−219 (lanes 7 and 8). <b>B</b>) Supershift EMSA, designed to identify specific protein interactions with −297/−163 radiolabeled probe. The reaction mixtures were incubated with pre-immune (lane 3), Sp-1(lane 4), β-catenin (lane 5), PU.1 (lane 6) and Ets-1(lane 7) antibodies before DNA-protein complexes were subjected to electrophoresis on 5% native PAGE (15 μg of protein/lane). Specific bands are shown with the filled arrows and super-shifted bands are shown with an open arrow.</p

    Identification of Ets-1 transcription factor as a protein interacting with −283/−252 oligonucleotide. A

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    <p>) Radiolabeled DNA probe encoding putative binding sites for Ets family of transcription factors (−283/−252) was incubated with 10–15<b> </b>μg of Hepa 1–6 and AML-12 nuclear cell lysate. The probe yielded similar band pattern with both the cell lysates. <b>B</b>) Competition EMSA was performed using labeled −283/−252 probe, Hepa 1–6 lysate and various competitor oligonucleotides at 50- and 200-fold molar excess. Competitor oligonucleotides used in EMSA are indicated above the figure. Specific bands are shown with arrows. <b>C</b>) Radiolabeled −283/−252 oligonucleotide was incubated with Hepa 1–6 nuclear lysate in the presence of either anti-PU.1 or anti-Ets-1 antibodies. DNA-protein complexes were resolved on 5% polyacrylamide gel and supershifted bands are shown with an open arrow. NE =  nuclear extract.</p

    A typical ChIP assay showing binding of Ets-1 and LEF/β-catenin to mouse PXR proximal promoter.

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    <p>Lane<b> 1</b> denotes PCR amplification of input DNA; lane<b> 2</b> shows PCR amplification of DNA immunoprecipitated using pre-immune serum (control IgG) and lanes<b> 3 & 6</b> represent PCR amplification of DNA immunoprecipitated by Ets-1 and β-catenin antibodies. No DNA was immunoprecipitated using PU.1 and Sp-1 antibodies (lanes<b> 4 & 5</b>).</p

    A list of forward (F) and reverse (R) primers used in the preparation of chimeric PXR promoter-luciferase reporter constructs, EMSA and ChIP analysis.

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    <p>A list of forward (F) and reverse (R) primers used in the preparation of chimeric PXR promoter-luciferase reporter constructs, EMSA and ChIP analysis.</p

    Deletion analysis of 1 kb proximal mouse PXR promoter by luciferase reporter gene assays.

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    <p>(<b>A</b>) Schematic representation of the 1 kb mouse PXR proximal promoter and its various serial deletions. The relative positions of different fragments are indicated. (<b>B</b>) Different PXR promoter luciferase reporter gene constructs were transiently co-transfected along with β-galactosidase into AML-12 cells. After24 hour of expression period, cell lysate was prepared and luciferase and β-galactosidase activities were determined. Levels of relative luciferase activities of different constructs are shown as fold change over the pGL3 basic vector. Data represent the mean ± SE of three different experiments. Asterisks (*) signify luciferase values that differed significantly from the pcDNA transfected cells (P<0.05 in Student's T-test).</p

    Deletion analysis of ∼5 kb mouse PXR promoter by luciferase reporter gene assays.

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    <p>(<b>A</b>) Schematic representation of the mouse PXR promoter and its various deletions constructs. (<b>B</b>) Plot showing relative luciferase activities of different constructs. The promoter-less basic luciferase vector and different PXR promoter-luciferase reporter gene constructs were transiently co-transfected into AML-12 cells along with β-galactosidase. After 24 hour of expression period, cell lysate was prepared and luciferase and β-galactosidase activities were determined. Luciferase values were normalized to β-galactosidase and expressed as fold change over the activity of basic luciferase vector. Data represent the mean ± SE of three independent experiments. Asterisks (*) signify luciferase values that differed significantly from the pcDNA transfected cells (P<0.05 in Student's T-test).</p
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