6-hydroxydopamine-induced nuclear factor-kappaB activation in PC12 cells22Abbreviations: 6-OHDA, 6-hydroxydopamine; EMSA, electrophoretic mobility shift assay; GSH, glutathione; IAP, inhibitory apoptosis protein; MAP, mitogen-activated protein; NAC, N-acetyl-cystein; NF-κB, nuclear factor-κB; and PD, Parkinson’s disease.

Thyrotropin (TSH) is a heterodimer consisting of α and β chains, and the β chain (TSHβ) is specific to TSH. The coexistence of two transcription factors, PIT1 and GATA2, is known to be essential for TSHβ expression. Using kidney-derived CV1 cells, we investigated the role of PIT1 in the expression of Tshb gene. GATA2 Zn finger domain, which is known to recognize GATA-responsive elements (GATA-REs), is essential for cooperation by PIT1. Transactivation of TSHβ promoter requires PIT1-binding site upstream to GATA-REs (PIT1-US), and the spacing between PIT1-US and GATA-REs strictly determines the cooperation between PIT1 and GATA2. Moreover, truncation of the sequence downstream to GATA-REs enabled GATA2 to transactivate the TSHβ promoter without PIT1. The deleted region (nt −82/−52) designated as a suppressor region (SR) was considered to inhibit transactivation by GATA2. The cooperation of PIT1 with GATA2 was not conventional synergism but rather counteracted SR-induced suppression (derepression). The minimal sequence for SR was mapped to the 9 bp sequence downstream to GATA-REs. Electrophoretic mobility shift assay suggested that some nuclear factor exists in CV1 cells, which binds with SR and this interaction was blocked by recombinant PIT1. Our study indicates that major activator for the TSHβ promoter is GATA2 and that PIT1 protects the function of GATA2 from the inhibition by SR-binding protein.

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Regulation of lactase–phlorizin hydrolase gene expression by the caudal-related homoeodomain protein Cdx-2

Biochemical Journal ◽

10.1042/bj3220833 ◽

1997 ◽

Vol 322 (3) ◽

pp. 833-838 ◽

Cited By ~ 115

Author(s):

Jesper T. TROELSEN ◽

Cathy MITCHELMORE ◽

Nikolaj SPODSBERG ◽

Anette M. JENSEN ◽

Ove NORÉN ◽

...

Keyword(s):

Gene Expression ◽

Small Intestine ◽

Binding Site ◽

Electrophoretic Mobility Shift Assay ◽

Gene Transcription ◽

Transcriptional Activity ◽

Nuclear Factor ◽

Mobility Shift ◽

Cis Element ◽

Mobility Shift Assay

Lactase–phlorizin hydrolase is exclusively expressed in the small intestine and is often used as a marker for the differentiation of enterocytes. The cis-element CE-LPH1 found in the lactase–phlorizin hydrolase promoter has previously been shown to bind an intestinal-specific nuclear factor. By electrophoretic mobility-shift assay it was shown that the factor Cdx-2 (a homoeodomain-protein related to caudal) binds to a TTTAC sequence in the CE-LPH1. Furthermore it was demonstrated that Cdx-2 is able to activate reporter gene transcription by binding to CE-LPH1. A mutation in CE-LPH1, which does not affect Cdx-2 binding, results in a higher transcriptional activity, indicating that the CE-LPH1 site contains other binding site(s) in addition to the Cdx-2-binding site.

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Cyclolinteinone, a sesterterpene from sponge Cacospongia linteiformis, prevents inducible nitric oxide synthase and inducible cyclo-oxygenase protein expression by blocking nuclear factor-κB activation in J774 macrophages

Biochemical Journal ◽

10.1042/bj3460793 ◽

2000 ◽

Vol 346 (3) ◽

pp. 793-798 ◽

Cited By ~ 9

Author(s):

Fulvio D'ACQUISTO ◽

Virginia LANZOTTI ◽

Rosa CARNUCCIO

Keyword(s):

Nitric Oxide ◽

Protein Expression ◽

Nuclear Translocation ◽

Inflammatory Diseases ◽

Nuclear Factor Κb ◽

Nuclear Factor ◽

Mobility Shift ◽

J774 Cells ◽

Cox 2 ◽

Mobility Shift Assay

We investigated the effect of cyclolinteinone, a sesterterpene from Caribbean sponge Cacospongia linteiformis, on inducible NO synthase (iNOS) and cyclo-oxygenase-2 (COX-2) protein expression in lipopolysaccharide (LPS)-stimulated J774 macrophages. Incubation of J774 cells with LPS (1 μg/ml) caused an increase of both iNOS and COX-2 protein expression, which was prevented in a concentration-dependent fashion by cyclolinteinone (12.5, 25 and 50 μM). Electrophoretic mobility-shift assay indicated that cyclolinteinone blocked the activation of nuclear factor-ĸB (NF-ĸB), a transcription factor necessary for either iNOS or COX-2 induction. Cyclolinteinone also blocked disappearance of IĸB-α from cytosolic fraction and nuclear translocation of NF-ĸB subunits p50 and p65. These results show that cyclolinteinone down-regulates iNOS and COX-2 protein expression by inhibiting NF-ĸB activation and suggest that it may represent a novel anti-inflammatory compound capable of controlling the excessive production of prostaglandins and nitric oxide occurring in several inflammatory diseases.

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Inhibition of the nuclear factor—κB activation with pyrrolidine dithiocarbamate attenuating inflammation and oxidative stress after experimental spinal cord trauma in rats

Journal of Neurosurgery Spine ◽

10.3171/spi.2004.1.3.0311 ◽

2004 ◽

Vol 1 (3) ◽

pp. 311-321 ◽

Cited By ~ 53

Author(s):

Giovanni La Rosa ◽

Salvatore Cardali ◽

Tiziana Genovese ◽

Alfredo Conti ◽

Rosanna Di Paola ◽

...

Keyword(s):

Spinal Cord ◽

Nuclear Factor Κb ◽

Pyrrolidine Dithiocarbamate ◽

Nuclear Factor ◽

Mobility Shift ◽

Content Type ◽

Cord Injury ◽

Mobility Shift Assay ◽

And Oxidative Stress ◽

Fine Print

Object. The nuclear factor—κB (NF-κB) is a transcription factor that plays a pivotal role in the induction of genes involved in physiological processes and in the response to inflammation. The authors of recent studies have demonstrated that NF-κB and oxidative stress contribute to secondary injury after impact-induced spinal cord injury (SCI) in the rat. Dithiocarbamates are antioxidants that are potent inhibitors of NF-κB. The authors postulated that pyrrolidine dithiocarbamate (PDTC) would attenuate NF-κB—related inflammatory and oxidative events that occur after SCI. Methods. Spinal cord injury was induced by the application of vascular clips (force of 50 g) to the dura mater after a four-level T5–8 laminectomy. The authors investigated the effects of PDTC (30 mg/kg administered 30 minutes before SCI and 6 hours after SCI) on the development of the inflammatory response associated with SCI in rats. Levels of myeloperoxidase activity were measured as an indicator of polymorphonuclear infiltration; malondialdehyde levels in the spinal cord tissue were determined as an indicator of lipid peroxidation. The following studies were performed: immunohistochemical analysis to assess levels of inducible nitric oxide synthase (iNOS), nitrotyrosine formation, poly([adenosine diphosphate]-ribose) polymerase (PARP) activity; Western blot analysis to determine cytoplasmic levels of inhibitory—κB-α (IκB-α); and electrophoretic mobility-shift assay to measure the level of DNA/NF-κB binding. The PDTC treatment exerted potent antiinflammatory effects with significant reduction of polymorphonuclear cell infiltration, lipid peroxidation, and iNOS activity. Furthermore, administration of PDTC reduced immunohistochemical evidence of formation of nitrotyrosine and PARP activation in the spinal cord section obtained in the SCI-treated rats. Additionally, PDTC treatment significantly prevented the activation of NF-κB (electrophoretic mobility-shift assay and immunoblot analysis). Conclusions. Overall, the results clearly demonstrate that PDTC-related prevention of the activation of NF-κB reduces the development of some secondary injury events after SCI. Therefore, inhibition of NF-κB may represent a novel approach in the treatment of SCIs.

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The orphan receptor hepatic nuclear factor 4 functions as a transcriptional activator for tissue-specific and hypoxia-specific erythropoietin gene expression and is antagonized by EAR3/COUP-TF1.

Molecular and Cellular Biology ◽

10.1128/mcb.15.4.2135 ◽

1995 ◽

Vol 15 (4) ◽

pp. 2135-2144 ◽

Cited By ~ 128

Author(s):

D L Galson ◽

T Tsuchiya ◽

D S Tendler ◽

L E Huang ◽

Y Ren ◽

...

Keyword(s):

Electrophoretic Mobility Shift Assay ◽

Electrophoretic Mobility ◽

Hela Cells ◽

Hormone Receptor ◽

Nuclear Factor ◽

Mobility Shift ◽

Response Elements ◽

Nuclear Extracts ◽

Hep3b Cells ◽

Mobility Shift Assay

The erythropoietin (Epo) gene is regulated by hypoxia-inducible cis-acting elements in the promoter and in a 3' enhancer, both of which contain consensus hexanucleotide hormone receptor response elements which are important for function. A group of 11 orphan nuclear receptors, transcribed and translated in vitro, were screened by the electrophoretic mobility shift assay. Of these, hepatic nuclear factor 4 (HNF-4), TR2-11, ROR alpha 1, and EAR3/COUP-TF1 bound specifically to the response elements in the Epo promoter and enhancer and, except for ROR alpha 1, formed DNA-protein complexes that had mobilities similar to those observed in nuclear extracts of the Epo-producing cell line Hep3B. Moreover, both anti-HNF-4 and anti-COUP antibodies were able to supershift complexes in Hep3B nuclear extracts. Like Epo, HNF-4 is expressed in kidney, liver, and Hep3B cells but not in HeLa cells. Transfection of a plasmid expressing HNF-4 into HeLa cells enabled an eightfold increase in the hypoxic induction of a luciferase reporter construct which contains the minimal Epo enhancer and Epo promoter, provided that the nuclear hormone receptor consensus DNA elements in both the promoter and the enhancer were intact. The augmentation by HNF-4 in HeLa cells could be abrogated by cotransfection with HNF-4 delta C, which retains the DNA binding domain of HNF-4 but lacks the C-terminal activation domain. Moreover, the hypoxia-induced expression of the endogenous Epo gene was significantly inhibited in Hep3B cells stably transfected with HNF-4 delta C. On the other hand, cotransfection of EAR3/COUP-TF1 and the Epo reporter either with HNF-4 into HeLa cells or alone into Hep3B cells suppressed the hypoxia induction of the Epo reporter. These electrophoretic mobility shift assay and functional experiments indicate that HNF-4 plays a critical positive role in the tissue-specific and hypoxia-inducible expression of the Epo gene, whereas the COUP family has a negative modulatory role.

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Nuclear factor-κB is constitutively activated in primitive human acute myelogenous leukemia cells

Blood ◽

10.1182/blood.v98.8.2301 ◽

2001 ◽

Vol 98 (8) ◽

pp. 2301-2307 ◽

Cited By ~ 519

Author(s):

Monica L. Guzman ◽

Sarah J. Neering ◽

Donna Upchurch ◽

Barry Grimes ◽

Dianna S. Howard ◽

...

Keyword(s):

Stem Cells ◽

Acute Myelogenous Leukemia ◽

Nuclear Factor Κb ◽

Myelogenous Leukemia ◽

Nuclear Factor ◽

Mobility Shift ◽

Human Cd34 ◽

Acute Myelogenous ◽

Mobility Shift Assay ◽

Gene Expression Studies

Abstract Human acute myelogenous leukemia (AML) is thought to arise from a rare population of malignant stem cells. Cells of this nature, herein referred to as leukemic stem cells (LSCs), have been documented for nearly all AML subtypes and appear to fulfill the criteria for stem cells in that they are self-renewing and give rise to the cells found in many leukemic populations. Because these cells are likely to be critical for the genesis and perpetuation of leukemic disease, the present studies sought to characterize unique molecular properties of the LSC population, with particular emphasis on the transcription factor, nuclear factor-κB (NF-κB). Previous experiments have shown that unstimulated human CD34+ progenitor cells do not express NF-κB. In contrast, primary AML CD34+ cells display readily detectable NF-κB activity as assessed by electrophoretic mobility shift assay and gene expression studies. Furthermore, detailed analyses of enriched AML stem cells (CD34+/CD38−/CD123+) indicate that NF-κB is also active in the LSC population. Given the expression of NF-κB in leukemic, but not normal primitive cells, the hypothesis that inhibition of NF-κB might induce leukemia-specific apoptosis was tested by treating primary cells with the proteasome inhibitor MG-132, a well-known inhibitor of NF-κB. Leukemic CD34+/CD38− cells displayed a rapid induction of cell death in response to MG-132, whereas normal CD34+/CD38− cells showed little if any effect. Taken together, these data indicate that primitive AML cells aberrantly express NF-κB and that the presence of this factor may provide unique opportunities to preferentially ablate LSCs.

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A fluorescence based non-radioactive electrophoretic mobility shift assay

Journal of Biotechnology ◽

10.1016/s0168-1656(00)00207-8 ◽

2000 ◽

Vol 78 (2) ◽

pp. 163-170 ◽

Cited By ~ 48

Author(s):

K Ruscher ◽

M Reuter ◽

D Kupper ◽

G Trendelenburg ◽

U Dirnagl ◽

...

Keyword(s):

Electrophoretic Mobility Shift Assay ◽

Electrophoretic Mobility ◽

Mobility Shift ◽

Mobility Shift Assay

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Human replication-dependent histone H3 genes are activated by a tandemly arranged pair of two CCAAT boxes

Biochemical Journal ◽

10.1042/bj20040502 ◽

2004 ◽

Vol 384 (2) ◽

pp. 317-326 ◽

Cited By ~ 5

Author(s):

Heiner KOESSLER ◽

Joerg KAHLE ◽

Christa BODE ◽

Detlef DOENECKE ◽

Werner ALBIG

Keyword(s):

Electrophoretic Mobility Shift Assay ◽

Electrophoretic Mobility ◽

Histone H3 ◽

Maximal Activity ◽

Significant Loss ◽

Tata Box ◽

Mobility Shift ◽

Nuclear Factor Y ◽

Binding Factor ◽

Mobility Shift Assay

We have analysed the transcriptional regulation of the human histone H3 genes using promoter deletion series, scanning mutagenesis, specific mutagenesis and electrophoretic mobility-shift assay experiments. The promoters of five of the six examined histone H3 genes showed near-maximal activity at lengths of 133–227 bp: H3/d 198 bp, H3/h 147 bp, H3/k 133 bp, H3/m 227 bp, H3/n 140 bp (exception H3/i). To search for functional cis-elements within these regions, we performed scanning mutagenesis of the two histone H3 promoters H3/k and H3/m. Mutagenesis revealed that the functional framework of the histone H3 promoters consists of a TATA box and two tandemly arranged CCAAT boxes in relatively fixed positions. Alterations of the distance between the CCAAT boxes and of the distance between the CCAAT boxes and the TATA box resulted in significant loss of activity. In electrophoretic mobility-shift assay experiments, the factor CBF (CCAAT-binding factor)/NF-Y (nuclear factor-Y) bound to isolated CCAAT boxes of the H3/k promoter. This suggests that an initiation complex is formed on the histone H3 promoter that has a defined structure and limited flexibility, consisting of two molecules of CBF/NF-Y and further (general or specific) transcription factors.

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