scholarly journals Increased Electrophoretic Mobility of Long-Type GATA-6 Transcription Factor upon Substitution of Its PEST Sequence

2014 ◽  
Vol 05 (13) ◽  
pp. 1032-1042
Author(s):  
Kanako Obayashi ◽  
Kayoko Takada ◽  
Kazuaki Ohashi ◽  
Ayako Ohashi-Kobayashi ◽  
Mayumi Nakanishi-Matsui ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Electrophoretic Mobility ◽  
Pest Sequence

Transcription factor binding study by capillary zone electrophoretic mobility shift assay

2003 ◽  
Vol 24 (12) ◽  
pp. 96-100 ◽  
Author(s):  
Zsolt Ronai ◽  
Yan Wang ◽  
Julia Khandurina ◽  
Paul Budworth ◽  
Maria Sasvari-Szekely ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Transcription Factor Binding ◽  
Binding Study ◽  
Mobility Shift ◽  
Factor Binding ◽  
Capillary Zone ◽  
Mobility Shift Assay

scholarly journals A detailed and radioisotope-free protocol for electrophoretic mobility shift assay (EMSA)

2021 ◽  
Author(s):  
NGUYEN HOAI NGUYEN
Keyword(s):  
Transcription Factor ◽  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Chromatin Immunoprecipitation ◽  
Direct Interaction ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Dna Fragment ◽  
Mobility Shift Assay

Abstract To comprehensively characterize the functions of a transcription factor (TF), it is required to analyze the interaction of this TF with its targeted loci. Several methods such as β-glucuronidase (GUS) or luciferase reporter, yeast one-hybrid (Y1H), chromatin-immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA) assays have been developed. Of these, EMSA is an in vitro method which can prove the direct interaction between TF and targeted DNA fragment. This protocol is to provide a detailed procedure for a safe EMSA assay (without using any radioisotope).

Identification of a novel region in the proximal promoter of the mouse renin gene critical for expression

2004 ◽  
Vol 286 (6) ◽  
pp. F1107-F1115 ◽  
Author(s):  
Li Pan ◽  
Craig A. Jones ◽  
Sean T. Glenn ◽  
Kenneth W. Gross
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Electrophoretic Mobility ◽  
Binding Sites ◽  
Promoter Activity ◽  
Transcription Factor Binding Sites ◽  
Proximal Promoter ◽  
Mobility Shift ◽  
Factor Binding ◽  
Renin Gene

An enhancer at −2.6 kb and a HOX·PBX-binding site at −60 bp have been demonstrated to be critical to expression of the mouse renin gene ( Ren-1 c) in As4.1 cells. In this report, we show that a region (−197 to −70) immediately 5′ to the HOX·PBX-binding site is also critical for Ren-1 c expression. Deletion of this region in a construct containing 4.1 kb of the Ren-1 c 5′-flanking sequence resulted in a 99% reduction in Ren-1 c promoter activity in As4.1 cells, suggesting the pivotal role for the region in the regulation of the mouse renin gene. Electrophoretic mobility shift and supershift assays have identified two nuclear factor I-binding sites and a Sp1/Sp3-binding site within the distal portion of the region (−197 to −103). Mutation of these three sites caused a 90% decrease in Ren-1 c promoter activity. Mutational analysis and electrophoretic mobility shift assays have also identified three additional transcription factor-binding sites within the region from −103 to −69, each of which contributes to high-level expression of Ren-1 c in As4.1 cells. Finally, we have shown that the Ren-1 c enhancer is the target for endothelin-1 (ET-1)-induced inhibition of Ren-1 c expression and the transcription factor-binding sites in the proximal promoter are required for the maximal ET-1 inhibitory effect.

scholarly journals Gene expression of the dibasic-pair cleaving enzyme NRD convertase (N-arginine dibasic convertase) is differentially regulated in the GH3 pituitary and Mat-Lu prostate cell lines

2000 ◽  
Vol 351 (3) ◽  
pp. 755-764
Author(s):  
Andrew G. WINTER ◽  
Adrian R. PIEROTTI
Keyword(s):  
Transcription Factor ◽  
Electrophoretic Mobility ◽  
Cell Lines ◽  
Binding Sites ◽  
Transcription Factor Binding Sites ◽  
Transcription Factor Binding ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Factor Binding ◽  
Electrophoretic Mobility Shift Assays

NRD convertase (N-arginine dibasic convertase, NRD-C) is a dibasic selective metalloprotease which cleaves on the N-terminal side of an arginine residue in a dibasic pair. Abundant in endocrine tissues, the highest levels are found in testis. The mechanism whereby NRD-C expression is regulated at the transcriptional level has been examined by reporter-gene assay and electrophoretic-mobility-shift assays. Analysis of the rat and human promoters show that they are highly conserved, containing a number of motifs which may correspond to transcription-factor binding sites. The rat promoter has been cloned into a luciferase reporter vector and analysed in a number of cell lines. Full functionality of the promoter is observed with 5′ deletions to 411bp upstream of the transcriptional start site in spermatid, prostate and pituitary cell lines. Further deletion to 101bp causes a complete loss of activity in spermatid and prostate lines. By contrast, GH3 pituitary cells display no reduction in promoter activity with deletion to 101bp of upstream sequence. A number of transcription-factor binding sites have been identified by electrophoretic-mobility-shift assays in the region 411–101; however, no differences in binding between the cell lines were observed.

scholarly journals Transcription Factor Nuclear Factor-Kappa B is Activated in Neurons after Focal Cerebral Ischemia

2000 ◽  
Vol 20 (3) ◽  
pp. 592-603 ◽  
Author(s):  
Diane Stephenson ◽  
Tinggui Yin ◽  
E. Barry Smalstig ◽  
Mei Ann Hsu ◽  
Jill Panetta ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cerebral Ischemia ◽  
Electrophoretic Mobility ◽  
Nuclear Factor Kappa B ◽  
Focal Cerebral Ischemia ◽  
Nuclear Factor ◽  
Shift Analysis ◽  
Gel Shift

Nuclear factor-kappa B (NF-kB) is a multisubunit transcription factor that when activated induces the expression of genes encoding acute-phase proteins, cell adhesion molecules, cell surface receptors, and cytokines. NF-kB is composed of a variety of protein subunits of which p50-and p65-kDa (RelA) are the most widely studied. Under resting conditions, these subunits reside in the cytoplasm as an inactive complex bound by inhibitor proteins, IkBα and IkBβ. On activation, IkB is phosphorylated by IkB kinase and ubiquitinated and degraded by the proteasome; simultaneously, the active heterodimer translocates to the nucleus where it can initiate gene transcription. In the periphery, NF-kB is involved in inflammation through stimulation of the production of inflammatory mediators. The role of NF-kB in the brain is unclear. In vitro, NF-kB activation can be either protective or deleterious. The role of NF-kB in ischemic neuronal cell death in vivo was investigated. Adult male rats were subjected to 2 hours of focal ischemia induced by middle cerebral artery occlusion (MCAO). At 2, 6, and 12 hours after reperfusion, the expression and transactivation of NF-kB in ischemic versus nonischemic cortex and striatum were determined by immunocytochemistry and by electrophoretic mobility gel-shift analysis. At all time points studied, p50 and p65 immunoreactivity was found exclusively in the nuclei of cortical and striatal neurons in the ischemic hemisphere. The contralateral nonischemic hemisphere showed no evidence of nuclear NF-kB immunoreactivity. Double immunofluorescence confirmed expression of p50 in nuclei of neurons. Increased NF-kB DNA-binding activity in nuclear extracts prepared from the ischemic hemisphere was further substantiated by electrophoretic mobility gel-shift analysis. Because the activation of NF-kB by many stimuli can be blocked by antioxidants in vitro, the effect of the antioxidant, LY341122, previously shown to be neuroprotective, on NF-kB activation in the MCAO model was evaluated. No significant activation of NF-kB was found by electrophoretic mobility gel-shift analysis in animals treated with LY341122. These results demonstrate that transient focal cerebral ischemia results in activation of NF-kB in neurons and supports previous observations that neuroprotective antioxidants may inhibit neuronal death by preventing the activation of NF-kB.

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