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Biotin RNA EMSA Kit from Pierce Med	06-06-2007 01:28 PM by admin »	4	716
DNA EMSA electrophoretic mobility shift assay darkwng	07-10-2006 04:56 AM by darkwng »	0	719
Emsa kaneja	06-18-2008 03:31 PM by admin »	1	26
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EMSA problem Balat	11-20-2006 07:39 AM by moleculardude »	1	744
I'm in EMSA Pain! Canalba	03-21-2007 04:58 AM by juvescott »	7	667
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Rna Emsa niligun	02-26-2007 03:05 AM by niligun »	0	502
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RNA EMSA - biotin-labeled RNA probe mayleung	02-27-2007 02:33 PM by frankcgl »	8	1404

EMSA with Proteins which bind non-specifically to DNA Hi All, Whilst i am not a total novice to EMSA and DNA binding assays i have come up against a brick wall with some of my current proteins of...
EMSA with PCR fragments Hi, Has anyone tried doing EMSA with PCR fragments of a promoter, instead of oligos? Thanks! T84
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Characterization of the human CIDEA promoter in fat cells. Related Articles

Characterization of the human CIDEA promoter in fat cells.

Int J Obes (Lond). 2008 Jul 8;

Authors: Pettersson AT, Laurencikiene J, Nordström EA, Stenson BM, van Harmelen V, Murphy C, Dahlman I, Rydén M

Background:Cell death-inducing DFFA (DNA fragmentation factor-alpha)-like effector A (CIDEA) is a protein that regulates lipolysis in human adipocytes through cross-talk involving tumor necrosis factor-alpha (TNF-alpha). TNF-alpha downregulates CIDEA mRNA although it is unclear whether this is mediated through transcriptional or post-transcriptional mechanisms. CIDEA has important metabolic effects in human fat cells and genetic variations in the human CIDEA gene have been correlated to the development of obesity. However, little is known about the factors regulating CIDEA expression in human adipocytes. We set out to describe the transcriptional control of human CIDEA.Methods:A 1.1-kb genomic fragment upstream of the transcriptional start site (TSS) of human CIDEA was cloned and deletion fragments were generated. Transcriptional activity of the promoter was analyzed by luciferase reporter assays in in vitro-differentiated human adipocytes. The effect of TNF-alpha was assessed in human adipocytes and murine 3T3-L1 cells transfected with deletion fragments of the CIDEA promoter. Protein-DNA interactions were analyzed by electrophoretic mobility shift assays (EMSA).Results:Basal transcriptional activity was found in a 97-bp region upstream of the TSS. We studied the effect of three common haplotypes in the promoter region but found no significant difference in transcriptional activity among them. Incubation of in vitro-differentiated human adipocytes as well as 3T3-L1 cells with TNF-alpha reduced the transcriptional activity of the human CIDEA promoter, demonstrating a direct effect on CIDEA transcription. EMSAs and mutational analysis indicated that this was mediated by a nuclear factor-kappaB (NF-kappaB) site at position -163/-151.Conclusion:We demonstrate that basal transcription of the human CIDEA gene is confined to the 97 first bases upstream of TSS and that TNF-alpha negatively regulates transcription of this gene, which at least in part involves NF-kappaB activation.International Journal of Obesity advance online publication, 8 July 2008; doi:10.1038/ijo.2008.101.

PMID: 18607384 [PubMed - as supplied by publisher]

The 'zinc knuckle' motif of Early B cell Factor is required for transcription... Related Articles

The 'zinc knuckle' motif of Early B cell Factor is required for transcriptional activation of B cell-specific genes.

Mol Immunol. 2008 Jul 5;

Authors: Fields S, Ternyak K, Gao H, Ostraat R, Akerlund J, Hagman J

Early B cell factor (EBF) is a critical regulator of B lymphocyte-specific gene transcription. EBF functions, in part, by binding to regulatory sites of genes required for the pre-B- and mature B cell receptors. These DNA targets include the promoters of the mb-1 and Vpreb1 genes that encode Ig-alpha and one of the components of surrogate light chain, respectively. The biochemical basis of DNA binding and gene activation by EBF is poorly understood. The DNA-binding domain (DBD) of EBF includes a putative zinc-binding motif (HX(3)CX(2)CX(5)C), which we have designated the 'Zn-knuckle'. The Zn-knuckle is required for binding of the mb-1 promoter site in EMSA, but it has not been demonstrated to be important for functional activities of EBF in B cells. Therefore, we expressed EBF with mutations in the Zn-knuckle motif or flanking sequences in plasmacytoma cells in which activation of endogenous mb-1 and Vpreb1 genes is dependent on EBF. EBF with mutations that prevent zinc coordination by the Zn-knuckle did not activate transcription of either target gene. Other mutations affected the sequence preference of DNA binding and differentially inhibited activation of these genes. Our results demonstrate the importance of the Zn-knuckle motif in EBF. These experiments also confirm that EBF can re-activate multiple genes of the early B cell program in plasmacytoma cells, which provide a useful cell-based assay for dissecting mechanisms involving EBF.

PMID: 18606452 [PubMed - as supplied by publisher]

Characterization and function of CREB homologue from Crassostrea ariakensis s... Related Articles

Characterization and function of CREB homologue from Crassostrea ariakensis stimulated by rickettsia-like organism.

Dev Comp Immunol. 2008 Jun 20;

Authors: Zhu B, Wu X

The cAMP response element-binding protein (CREB) is a transcription factor that plays important roles in cellular growth, proliferation and survival. Here, we report that a homologue of CREB transcription factor, Ca-CREB, was identified and functionally characterized in oyster, Crassostrea ariakensis. The full-length cDNA consists of 1397bp with an ORF encoding a 39.3kDa protein. Amino acid sequence analysis revealed that Ca-CREB shares conserved signature motifs with other CREB proteins. Ca-CREB was ubiquitously and constitutively expressed in oyster, and the expression level in hemocytes was higher than that in other tissues. The expression level of Ca-CREB was not modified after RLO stimulation, while tumor necrosis factor-alpha (TNF-alpha) expression was increased obviously, which was revealed by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Electrophoretic mobility shift assay (EMSA) and Western blotting showed that recombinant CREB proteins specifically bind the consensus CREB binding site, and DNA-binding activity and phosphorylation of Ca-CREB were induced by RLO. These results suggest that Ca-CREB is a CREB homologue and may be involved in immune responses against RLO.

PMID: 18606451 [PubMed - as supplied by publisher]

[Inhibition of NF-kappaB Through IkappaBalpha Transfection Affects Invasion o... Related Articles

[Inhibition of NF-kappaB Through IkappaBalpha Transfection Affects Invasion of Human Lung Cancer Cell Line A549.]

Ai Zheng. 2008 Jul;27(7):710-5

Authors: Zhang J, Xu YJ, Xiong WN, Zhang ZX, DU CL, Qiao LF, Ni W, Chen SX

BACKGROUND & OBJECTIVE: Recent studies have shown that activation of nuclear factor-kappaB(NF-kappaB) can regulate the invasion and metastasis of cancer cells. The present study was to investigate inhibition of NF-kappaB activity on invasion of human lung cancer cell line A549 and the possible mechanism. METHODS: The recombinant plasmid pcDNA3.1(+)/IkappaBalpha, expressing the alpha isoform (IkappaBalpha) of the NF-kappaB inhibitor, was constructed. A549 cells were cultured in vitro and divided into non-transfection group, pcDNA3.1(+) transfected group and pcDNA3.1(+)/IkappaBalpha transfected group. The expression of IkappaBalpha was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The activity of NF-kappaB was determined by electrophoretic mobility shift assay (EMSA). Invasion of A549 cells was assessed by transwell chamber assay. The expression of MMP-2 and MMP-9 was detected by RT-PCR and gelatin zymography. RESULTS: Plasmid pcDNA3.1(+)/IkappaBalpha was successfully constructed and expressed in A549 cells. The activity of NF-kappaB, the number of invasive cells, the activity of MMP-2 and MMP-9 of A549 cells in pcDNA3.1(+)/IkappaBalpha transfected group were significantly lower than those in non-transfection group and pcDNA3.1(+) transfected group (all P<0.05). CONCLUSION: Transfection of IkappaBalpha can inhibit NF-kappaB activity, thus inhibit cell invasion of A549, which may be through the down-regulation of MMP-2 and MMP-9 expressions.

PMID: 18606063 [PubMed - in process]

[Effects of SB203580 and WP631 on Smad Signal Transduction Pathway in Lung Fi... Related Articles

[Effects of SB203580 and WP631 on Smad Signal Transduction Pathway in Lung Fibroblasts after Irradiation.]

Ai Zheng. 2008 Jul;27(7):698-702

Authors: Li Y, Song LW, Peng RY, Wang DW, Jin MH, Gao YB, Ma JJ

BACKGROUND & OBJECTIVE: Radiation pulmonary fibrosis (RPF) is characterized by fibroblast proliferation and excessive accumulation of extracellular matrix (ECM). Transforming growth factor beta (TGFbeta) is a switch factor in the initiation and development of RPF and serves as a therapeutic target. Blocking TGFbeta1 signal transduction pathway might alleviate RPF. This study was to investigate the effects of two Smad pathway inhibitors, SB203580 and WP631, on Smad signal transduction pathway in human lung fibroblasts (HLFs) after irradiation. METHODS: HLFs were pretreated with 25 mumol/L SB203580 or 5 nmol/L WP631, then irradiated with 3 Gy 60Co gamma rays and stimulated by 10 mug/L TGFbeta1. The transcriptional activity of SP1 and AP1 were measured using electrophoretic mobility shift assay (EMSA). Expressions of Smad3, Smad4, Smad7, p-Smad3 and P21(WAF1/CIP1) were detected by Western blot. The expression of type Iplasminogen activator inhibitor (PAI-I) was detected by immunohistochemical staining The cell cycle was measured by FACS. RESULTS: After irradiation. with 3 Gy gamma rays and stimulation by TGFbeta1, HLFs pre-incubated with SB203580 or WP631 were increased in G2-M phase and decreased in S phase as compared with cells without pretreatment. p21(WAF1/CIP1) and p-Smad3 were decreased in HLFs pretreated with SB203580, while PAI-1 was decreased in HLFs pretreated with WP631. Furthermore, the transcriptional activity of SP1 and AP1 was inhibited by WP631. CONCLUSIONS: SB203580 and WP631 can abrogate excessive proliferation, expressions of p21(WAF1/CIP1) and PAI-1 induced by gamma rays and TGFbeta1 in HLFs through blocking Smad signal transduction pathway.

PMID: 18606061 [PubMed - in process]