Rapid qualitative evaluation of DNA transcription factor NF‐κB by microchip electrophoretic mobility shift assay in mammalian cells

Inoue, Sonoko; Kaji, Noritada; Kataoka, Masatoshi; Shinohara, Yasuo; Okamoto, Yukihiro; Tokeshi, Manabu; Baba, Yoshinobu

doi:10.1002/elps.201100261

Cited by 6 publications

(6 citation statements)

References 29 publications

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“…Our study largely focused on hepatocytes, since the signal from whole-liver tissue ChIP is likely predominantly contributed by hepatocytes in contrast to non-parenchymal cells. Emerging techniques such as micro-ChIP and single cell ChIP methods potentially enable study of NF-κB genome-wide binding responses in non-parenchymal cells 103–106 .…”

Section: Discussionmentioning

confidence: 99%

A novel comparative pattern count analysis reveals a chronic ethanol-induced dynamic shift in immediate early NF-κB genome-wide promoter binding during liver regeneration

et al. 2016

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Liver regeneration after partial hepatectomy is a clinically important process that is impaired by adaptation to chronic alcohol intake. We focused on the initial time points following partial hepatectomy (PHx) to analyze genome-wide binding activity of NF-κB, a key immediate early regulator. We investigated the effect of chronic alcohol intake on immediate early NF-κB genome-wide localization, in the adapted state as well as in response to partial hepatectomy, using chromatin immunoprecipitation followed by promoter microarray analysis. We found many ethanol-specific NF-κB binding target promoters in the ethanol-adapted state, corresponding to regulation of biosynthetic processes, oxidation-reduction and apoptosis. Partial hepatectomy induced a diet-independent shift in NF-κB binding loci relative to the transcription start sites. We employed a novel pattern count analysis to exhaustively enumerate and compare the number of promoters corresponding to the temporal binding patterns in ethanol and pair-fed control groups. The highest pattern count corresponded to promoters with NF-κB binding exclusively in the ethanol group at 1h post PHx. This set was associated with regulation of cell death, response to oxidative stress, histone modification, mitochondrial function, and metabolic processes. Integration with the global gene expression profiles to identify putative transcriptional consequences of NF-κB binding patterns revealed that several of ethanol-specific 1h binding targets showed ethanol-specific differential expression through 6h post PHx. Motif analysis yielded co-incident binding loci for STAT3, AP-1, CREB, C/EBP-β, PPAR-γ and C/EBP-α, likely participating in co-regulatory modules with NF-κB in shaping the immediate early response to PHx. We conclude that adaptation to chronic ethanol intake disrupts the NF-κB promoter binding landscape with consequences for the immediate early gene regulatory response to the acute challenge of PHx.

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Section: Discussionmentioning

confidence: 99%

A novel comparative pattern count analysis reveals a chronic ethanol-induced dynamic shift in immediate early NF-κB genome-wide promoter binding during liver regeneration

et al. 2016

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“…The single-channel, two-port architecture is well-suited for scale-up into, for example, a dense array of separation channels. We see this approach as broadly relevant to high-throughput targeted proteomics and migration shift screening. − …”

Section: Discussionmentioning

confidence: 99%

Use of Polyacrylamide Gel Moving Boundary Electrophoresis to Enable Low-Power Protein Analysis in a Compact Microdevice

Duncombe

Herr

2012

Anal. Chem.

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In designing a protein electrophoresis platform composed of a single-inlet, single-outlet microchannel powered solely by voltage control (no pumps, values, injectors), we adapted the original protein electrophoresis format-moving boundary electrophoresis (MBE)-to a high-performance, compact microfluidic format. Key to the microfluidic adaptation is minimization of injection dispersion during sample injection. To reduce injection dispersion, we utilize a photopatterned free-solution-polyacrylamide gel (PAG) stacking interface at the head of the MBE microchannel. The nanoporous PAG molecular sieve physically induces a mobility shift that acts to enrich and sharpen protein fronts as proteins enter the microchannel. Various PAG configurations are characterized, with injection dispersion reduced by up to 85%. When employed for analysis of a model protein sample, microfluidic PAG MBE baseline-resolved species in 5 s and in a separation distance of less than 1 mm. PAG MBE thus demonstrates electrophoretic assays with minimal interfacing and sample handling, while maintaining separation performance. Owing to the short separation lengths needed in PAG MBE, we reduced the separation channel length to demonstrate an electrophoretic immunoassay powered with an off-the-shelf 9 V battery. The electrophoretic immunoassay consumed less than 3 μW of power and was completed in 30 s. To our knowledge, this is the lowest voltage and lowest power electrophoretic protein separation reported. Looking forward, we see the low-power PAG MBE as a basis for highly multiplexed protein separations (mobility shift screening assays) as well as for portable low-power diagnostic assays.

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“…Several classes of quantitative affinity-based microfluidic EMSAs including immunoassays (IAs), affinity EMSAs, dragtag-based EMSAs, and other were elaborated by Pan et al (2014) . A separation technique for DNA–protein complex which called microchip electrophoretic mobility shift assay (μEMSA), based on EMSA by microchip electrophoresis was developed by Inoue et al (2011) . The performance of EMSA linked with nanoparticle–aptamer conjugates (NP-EMSA) was improved over the traditional EMSA ( Wang and Reed, 2012 ).…”

Section: Conventional Methods For Detecting Protein–dna Interaction Amentioning

confidence: 99%

Choosing a suitable method for the identification of replication origins in microbial genomes

2015

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As the replication of genomic DNA is arguably the most important task performed by a cell and given that it is controlled at the initiation stage, the events that occur at the replication origin play a central role in the cell cycle. Making sense of DNA replication origins is important for improving our capacity to study cellular processes and functions in the regulation of gene expression, genome integrity in much finer detail. Thus, clearly comprehending the positions and sequences of replication origins which are fundamental to chromosome organization and duplication is the first priority of all. In view of such important roles of replication origins, tremendous work has been aimed at identifying and testing the specificity of replication origins. A number of computational tools based on various skew types have been developed to predict replication origins. Using various in silico approaches such as Ori-Finder, and databases such as DoriC, researchers have predicted the locations of replication origins sites for thousands of bacterial chromosomes and archaeal genomes. Based on the predicted results, we should choose an effective method for identifying and confirming the interactions at origins of replication. Here we describe the main existing experimental methods that aimed to determine the replication origin regions and list some of the many the practical applications of these methods.

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Rapid qualitative evaluation of DNA transcription factor NF‐κB by microchip electrophoretic mobility shift assay in mammalian cells

Cited by 6 publications

References 29 publications

A novel comparative pattern count analysis reveals a chronic ethanol-induced dynamic shift in immediate early NF-κB genome-wide promoter binding during liver regeneration

A novel comparative pattern count analysis reveals a chronic ethanol-induced dynamic shift in immediate early NF-κB genome-wide promoter binding during liver regeneration

Use of Polyacrylamide Gel Moving Boundary Electrophoresis to Enable Low-Power Protein Analysis in a Compact Microdevice

Choosing a suitable method for the identification of replication origins in microbial genomes

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