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Lewis A Chodosh - One of the best experts on this subject based on the ideXlab platform.
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mobility shift DNA Binding Assay using gel electrophoresis
Current protocols in pharmacology, 2001Co-Authors: Stephen Buratowski, Lewis A ChodoshAbstract:DNA-Binding Assay using nondenaturing polyacrylamide gel electrophoresis (PAGE) provides a simple, rapid, and extremely sensitive method for detecting sequence-specific DNA-Binding proteins. Proteins that bind specifically to an end-labeled DNA fragment retard the mobility of the fragment during electrophoresis, resulting in discrete bands corresponding to the individual protein-DNA complexes. The Assay described in this unit can be used to test Binding of purified proteins or of uncharacterized factors found in crude extracts. This Assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and Binding specificity of DNA-Binding proteins. Three additional protocols describe a competition Assay using unlabeled competitor DNA, an antibody supershift Assay, and multicomponent gel shift Assays.
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mobility shift DNA Binding Assay using gel electrophoresis
Current protocols in molecular biology, 1996Co-Authors: Stephen Buratowski, Lewis A ChodoshAbstract:The DNA-Binding Assay using nondenaturing polyacrylamide gel electrophoresis (PAGE) provides a simple, rapid, and extremely sensitive method for detecting sequence-specific DNA-Binding proteins. Proteins that bind specifically to an end-labeled DNA fragment retard the mobility of the fragment during electrophoresis, resulting in discrete bands corresponding to the individual protein-DNA complexes. The Assay described in this unit can be used to test Binding of purified proteins or of uncharacterized factors found in crude extracts. This Assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and Binding specificity of DNA-Binding proteins. Three additional protocols describe a competition Assay using unlabeled competitor DNA, an antibody supershift Assay, and multicomponent gel shift Assays.
Stephen Buratowski - One of the best experts on this subject based on the ideXlab platform.
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mobility shift DNA Binding Assay using gel electrophoresis
Current protocols in pharmacology, 2001Co-Authors: Stephen Buratowski, Lewis A ChodoshAbstract:DNA-Binding Assay using nondenaturing polyacrylamide gel electrophoresis (PAGE) provides a simple, rapid, and extremely sensitive method for detecting sequence-specific DNA-Binding proteins. Proteins that bind specifically to an end-labeled DNA fragment retard the mobility of the fragment during electrophoresis, resulting in discrete bands corresponding to the individual protein-DNA complexes. The Assay described in this unit can be used to test Binding of purified proteins or of uncharacterized factors found in crude extracts. This Assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and Binding specificity of DNA-Binding proteins. Three additional protocols describe a competition Assay using unlabeled competitor DNA, an antibody supershift Assay, and multicomponent gel shift Assays.
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mobility shift DNA Binding Assay using gel electrophoresis
Current protocols in molecular biology, 1996Co-Authors: Stephen Buratowski, Lewis A ChodoshAbstract:The DNA-Binding Assay using nondenaturing polyacrylamide gel electrophoresis (PAGE) provides a simple, rapid, and extremely sensitive method for detecting sequence-specific DNA-Binding proteins. Proteins that bind specifically to an end-labeled DNA fragment retard the mobility of the fragment during electrophoresis, resulting in discrete bands corresponding to the individual protein-DNA complexes. The Assay described in this unit can be used to test Binding of purified proteins or of uncharacterized factors found in crude extracts. This Assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and Binding specificity of DNA-Binding proteins. Three additional protocols describe a competition Assay using unlabeled competitor DNA, an antibody supershift Assay, and multicomponent gel shift Assays.
Fredric E Wondisford - One of the best experts on this subject based on the ideXlab platform.
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thyrotropin releasing hormone regulation of human tshb expression role of a pituitary specific transcription factor pit 1 ghf 1 and potential interaction with a thyroid hormone inhibitory element cis acting element thyrotroph DNA Binding Assay trans acting factor
2016Co-Authors: Hans Jurgen Steinfelder, Sally Radovick, Bruce D. Weintraub, Peter Hauser, Yuko Nakayama, John H Mcclaskey, Fredric E WondisfordAbstract:Regulation of human thyrotropin ,3 subunit gene (TSHB) expression by thyrotropin-releasing hormone (TRH) was examined in a clonal rat pituitary-cell line (GH3). Transient expression studies were done with various 5'- flanking DNA sequences of TSHB coupled to reporter gene chloramphenicol acetyltransferase. Deletion analysis defined two discrete regions (-128 to -92 base pairs and -28 to +8 base pairs) that each mediated an --2-fold TRH induction. The upstream site contains a DNA sequence with close homology to the DNA-Binding site for a pituitary-specific transcriptional factor Pit-1/GHF-1. DNAse I footprinting analysis of mouse thyrotropic tumor extract as well as DNA-transfection studies using an expression vector containing an N-terminal deletion of Pit-1/GHF-1 cDNA suggest that Pit-1/GHF-1 or a closely related protein in the thyrotroph mediates TRH responsiveness of this gene. In addition, the downstream site overlaps with the recently characterized thyroid hormone-inhibitory element of TSHB. In fact, deletion of DNA sequences important in thyroid hormone-receptor Binding (c-erbAB/c-ERBA2) from +3 to +8 base pairs, significantly reduced (30%) TRH responsiveness. The location of a TRH-stimulatory element near a thyroid hormone-inhibitory element may allow for fine control of TSHB expression in vivo. Thyroid-stimulating hormone like other pituitary glycopro- tein hormones contains two dissimilar noncovalently linked subunits, a and f8. The major negative regulator of the
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a detailed functional and structural analysis of a major thyroid hormone inhibitory element in the human thyrotropin beta subunit gene
Journal of Biological Chemistry, 1991Co-Authors: Donald L. Bodenner, Melisa A Mroczynski, Sally Radovick, Bruce D. Weintraub, Fredric E WondisfordAbstract:Abstract The first exon of the human thyrotropin-beta (hTSH beta) gene has been demonstrated in our laboratory to contain a major thyroid hormone inhibitory element. In order to characterize fully this element, we have performed a detailed functional and structural scanning mutational analysis of this element. Various -1192 to +37 (base pairs) bp fragments of the hTSH beta gene containing consecutive five deoxythymidine substitution mutations of the first exon were inserted into a luciferase reporter plasmid and transiently transfected into human embryonal cells (293) and stably transfected into rat pituitary cells (GH3). Two domains (domain 1 and 2) were identified by scanning mutations that were essential for function of the thyroid hormone inhibitory element: +3 to +13 bp and +28 to +37 bp. Biotinylated DNA fragments containing -12 to +43 bp of the hTSH beta gene and the identical scanning mutations demonstrate that in vitro synthesized c-erbA-beta Binding is disrupted as much as 95% by mutations from -3 to +17 bp and to a lesser extent (20-30%) by mutations from +23 to +27 bp and from +33 to +43 bp. Domain 1 displayed a higher affinity for c-erbA-beta than domain 2 in avidin-biotin complex DNA-Binding and gel-mobility Assays. Using increasing amounts of in vitro synthesized c-erbA-beta, we were unable to demonstrate more than one protein-DNA complex in gel-mobility Assays. However, using the avidin-biotin complex DNA-Binding Assay and the cross-linking reagent, 1,6-bismaleimidohexane, we were able to demonstrate thyroid hormone receptor dimer formation on domain 1 but not to any significant extent on domain 2. In conclusion, functional and DNA-Binding studies suggest that the thyroid hormone receptor binds to two distinct regions in the first exon of the hTSH beta gene. The upstream site (domain 1) binds c-erbA-beta with higher affinity and is capable of Binding c-erbA-beta as a dimer under some conditions, while the downstream site (domain 2) appears to bind a single molecule of c-erbA-beta with lower affinity. These results suggest that thyroid hormone receptor, Binding to at least two sites in the first exon, act in conjunction to mediate T3 inhibition of hTSH beta expression.
Gautam Sethi - One of the best experts on this subject based on the ideXlab platform.
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first evidence that γ tocotrienol inhibits the growth of human gastric cancer and chemosensitizes it to capecitabine in a xenograft mouse model through the modulation of nf κb pathway
Clinical Cancer Research, 2012Co-Authors: Kanjoormana Aryan Manu, Muthu K Shanmugam, Lalitha Ramachandran, Chee Wai Fong, Alan Prem Kumar, Patrick Tan, Gautam SethiAbstract:Purpose: Because of poor prognosis and development of resistance against chemotherapeutic drugs, the existing treatment modalities for gastric cancer are ineffective. Hence, novel agents that are safe and effective are urgently needed. Whether γ-tocotrienol can sensitize gastric cancer to capecitabine in vitro and in a xenograft mouse model was investigated. Experimental Design: The effect of γ-tocotrienol on proliferation of gastric cancer cell lines was examined by mitochondrial dye uptake Assay, apoptosis by esterase staining, NF-κB activation by DNA-Binding Assay, and gene expression by Western blotting. The effect of γ-tocotrienol on the growth and chemosensitization was also examined in subcutaneously implanted tumors in nude mice. Results: γ-Tocotrienol inhibited the proliferation of various gastric cancer cell lines, potentiated the apoptotic effects of capecitabine, inhibited the constitutive activation of NF-κB, and suppressed the NF-κB–regulated expression of COX-2, cyclin D1, Bcl-2, CXCR4, VEGF, and matrix metalloproteinase-9 (MMP-9). In a xenograft model of human gastric cancer in nude mice, we found that administration of γ-tocotrienol alone (1 mg/kg body weight, intraperitoneally 3 times/wk) significantly suppressed the growth of the tumor and this effect was further enhanced by capecitabine. Both the markers of proliferation index Ki-67 and for microvessel density CD31 were downregulated in tumor tissue by the combination of capecitabine and γ-tocotrienol. As compared with vehicle control, γ-tocotrienol also suppressed the NF-κB activation and the expression of cyclin D1, COX-2, intercellular adhesion molecule-1 (ICAM-1), MMP-9, survivin, Bcl-xL, and XIAP. Conclusions: Overall our results show that γ-tocotrienol can potentiate the effects of capecitabine through suppression of NF-κB–regulated markers of proliferation, invasion, angiogenesis, and metastasis. Clin Cancer Res; 18(8); 2220–9. ©2012 AACR .
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morin 3 5 7 2 4 pentahydroxyflavone abolishes nuclear factor κb activation induced by various carcinogens and inflammatory stimuli leading to suppression of nuclear factor κb regulated gene expression and up regulation of apoptosis
Clinical Cancer Research, 2007Co-Authors: Sunil K Manna, Bharat B Aggarwal, Rishi S Aggarwal, Gautam Sethi, Govindarajan T RameshAbstract:Purpose: Morin is a flavone that exhibits antiproliferative, antitumor, and anti-inflammatory effects through a mechanism that is not well understood. Because of the role of transcription factor nuclear factor-κB (NF-κB) in the control of cell survival, proliferation, tumorigenesis, and inflammation, we postulated that morin mediates its effects by modulating NF-κB activation. Experimental Design: We investigated the effect of morin on NF-κB pathway activated by inflammatory agents, carcinogens, and tumor promoters. The effect of this flavone on expression of NF-κB–regulated gene products involved in cell survival, proliferation, and invasion was also examined. Results: We showed by DNA-Binding Assay that NF-κB activation induced by tumor necrosis factor (TNF), phorbol 12-myristate 13-acetate, lipopolysaccharide, ceramide, interleukin-1, and H 2 O 2 was suppressed by morin; the suppression was not cell type specific. The suppression of NF-κB by morin was mediated through inhibition of IκBα (inhibitory subunit of NF-κB) kinase, leading to suppression of phosphorylation and degradation of IκBα and consequent p65 nuclear translocation. Morin also inhibited the NF-κB–dependent reporter gene expression activated by TNF, TNF receptor (TNFR) 1, TNFR1-associated death domain, TNFR-associated factor 2, NF-κB–inducing kinase, IκB kinase, and the p65 subunit of NF-κB. NF-κB–regulated gene products involved in cell survival [inhibitor of apoptosis (IAP) 1, IAP2, X chromosome-linked IAP, Bcl-xL, and survivin], proliferation (cyclin D1 and cyclooxygenase-2), and invasion (matrix metalloproteinase-9) were down-regulated by morin. These effects correlated with enhancement of apoptosis induced by TNF and chemotherapeutic agents. Conclusion: Overall, our results indicate that morin suppresses the activation of NF-κB and NF-κB–regulated gene expression, leading to enhancement of apoptosis. This may provide the molecular basis for the ability of morin to act as an anticancer and anti-inflammatory agent.
Bharat B Aggarwal - One of the best experts on this subject based on the ideXlab platform.
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morin 3 5 7 2 4 pentahydroxyflavone abolishes nuclear factor κb activation induced by various carcinogens and inflammatory stimuli leading to suppression of nuclear factor κb regulated gene expression and up regulation of apoptosis
Clinical Cancer Research, 2007Co-Authors: Sunil K Manna, Bharat B Aggarwal, Rishi S Aggarwal, Gautam Sethi, Govindarajan T RameshAbstract:Purpose: Morin is a flavone that exhibits antiproliferative, antitumor, and anti-inflammatory effects through a mechanism that is not well understood. Because of the role of transcription factor nuclear factor-κB (NF-κB) in the control of cell survival, proliferation, tumorigenesis, and inflammation, we postulated that morin mediates its effects by modulating NF-κB activation. Experimental Design: We investigated the effect of morin on NF-κB pathway activated by inflammatory agents, carcinogens, and tumor promoters. The effect of this flavone on expression of NF-κB–regulated gene products involved in cell survival, proliferation, and invasion was also examined. Results: We showed by DNA-Binding Assay that NF-κB activation induced by tumor necrosis factor (TNF), phorbol 12-myristate 13-acetate, lipopolysaccharide, ceramide, interleukin-1, and H 2 O 2 was suppressed by morin; the suppression was not cell type specific. The suppression of NF-κB by morin was mediated through inhibition of IκBα (inhibitory subunit of NF-κB) kinase, leading to suppression of phosphorylation and degradation of IκBα and consequent p65 nuclear translocation. Morin also inhibited the NF-κB–dependent reporter gene expression activated by TNF, TNF receptor (TNFR) 1, TNFR1-associated death domain, TNFR-associated factor 2, NF-κB–inducing kinase, IκB kinase, and the p65 subunit of NF-κB. NF-κB–regulated gene products involved in cell survival [inhibitor of apoptosis (IAP) 1, IAP2, X chromosome-linked IAP, Bcl-xL, and survivin], proliferation (cyclin D1 and cyclooxygenase-2), and invasion (matrix metalloproteinase-9) were down-regulated by morin. These effects correlated with enhancement of apoptosis induced by TNF and chemotherapeutic agents. Conclusion: Overall, our results indicate that morin suppresses the activation of NF-κB and NF-κB–regulated gene expression, leading to enhancement of apoptosis. This may provide the molecular basis for the ability of morin to act as an anticancer and anti-inflammatory agent.
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protein farnesyltransferase inhibitor sch 66336 abolishes nf κb activation induced by various carcinogens and inflammatory stimuli leading to suppression of nf κb regulated gene expression and up regulation of apoptosis
Journal of Biological Chemistry, 2004Co-Authors: Yasunari Takada, Fadlo R Khuri, Bharat B AggarwalAbstract:Abstract Ras farnesyltransferase inhibitor (FTI) exhibit antiproliferative and antiangiogenic effects through a mechanism that is poorly understood. Because of the known role of Ras in the activation of transcription factor NF-κB and because NF-κB-regulated genes can control cell survival and angiogenesis, we postulated that FTI mediates its effects in part by modulating NF-κB activation. Therefore, in the present study we investigated the effect of FTI, SCH 66336, on NF-κB and NF-κB-regulated gene expression activated by a variety of inflammatory and carcinogenic agents. We demonstrate by DNA-Binding Assay that NF-κB activation induced by tumor necrosis factor (TNF), phorbol 12-myristate 13-acetate, cigarette smoke, okadaic acid, and H2O2 was completely suppressed by SCH 66336; the suppression was not cell type-specific. This FTI suppressed the activation of IκBα kinase (IKK), thus abrogating the phosphorylation and degradation of IκBα. Additionally, TNF-activated Ras and SCH 66336 inhibited the activation. Also, overexpression of Ras (V12) enhanced TNF-induced NF-κB activation, and adenoviral dominant-negative Ras (N17) suppressed the activation, thus suggesting the critical role of Ras in TNF signaling. SCH 66336 also inhibited the NF-κB-dependent reporter gene expression activated by TNF, TNFR1, TRADD, TRAF2, NIK, and IKK but not that activated by the p65 subunit of NF-κB. The TNF-induced NF-κB-regulated gene products cyclin D1, COX-2, MMP-9, survivin, IAP1, IAP2, XIAP, Bcl-2, Bfl-1/A1, TRAF1, and FLIP were all down-regulated by SCH 66336, which potentiated apoptosis induced by TNF and doxorubicin. Overall, our results indicate that SCH 66336 inhibited activation of NF-κB and NF-κB-regulated gene expressions induced by carcinogens and inflammatory stimuli, which may provide a molecular basis for the ability of SCH 66336 to suppress proliferation and angiogenesis.