Exonuclease-mediated ELISA-like assay for detecting DNA-binding activity of transcription factors: measurement of activated NF-κB

Wang, Jinke; Li, Min L.; Dong, Honglin; Chen, Qixin

doi:10.2144/000112218

Cited by 10 publications

(7 citation statements)

References 56 publications

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“…# 07-200-585) at a concentration of 25 pmol in a 100 μl volume per each well in oligonucleotide binding buffer (50 mM Na 3 PO 4 , pH 8.5, 1 mM EDTA) and washed extensively (Supplemental Figure 1S-C). Five pmol of the antisense oligo was added and a dsDNA was formed as in [50]. Plate-bound DNA was incubated with up to 30 ng/well total of homodimers or heterdimers of the nuclear receptors (PPARα, RXRα or LXRα) (if heterodimers like PPARα+LXRα, then in equal concentrations) for 20 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

A Phosphatidic Acid (PA) conveyor system of continuous intracellular transport from cell membrane to nucleus maintains EGF receptor homeostasis

et al. 2016

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The intracellular concentration of the mitogen phosphatidic acid (PA) must be maintained at low levels until the need arises for cell proliferation. How temporal and spatial trafficking of PA affects its target proteins in the different cellular compartments is not fully understood. We report that in cancer cells, PA cycles back and forth from the cellular membrane to the nucleus, affecting the function of epidermal growth factor (EGF), in a process that involves PPARα/LXRα signaling. Upon binding to its ligand, EGF receptor (EGFR)-initiated activation of phospholipase D (PLD) causes a spike in intracellular PA production that forms vesicles transporting EGFR from early endosomes (EEA1 marker) and prolonged internalization in late endosomes and Golgi (RCAS marker). Cells incubated with fluorescent-labeled PA (NBD-PA) show PA in “diffuse” locations throughout the cytoplasm, punctae (small, <0.1 μm) vesicles) and large (>0.5 μm) vesicles that co-localize with EGFR. We also report that PPARα/LXRα form heterodimers that bind to new Responsive Elements (RE) in the EGFR promoter. Nuclear PA enhances EGFR expression, a role compatible with the mitogenic ability of the phospholipid. Newly made EGFR is packaged into PA recycling vesicles (Rab11 marker) and transported back to the cytoplasm and plasma membrane. However, a PLD+PA combination impedes binding of PPARα/LXRα to the EGFR promoter. Thus, if PA levels inside the nucleus reach a certain threshold (>100 nM) PA outcompetes the nuclear receptors and transcription is inhibited. This new signaling function of PLD-PA targeting EGFR trafficking and biphasically modulating its transcription, could explain cell proliferation initiation and its maintenance in cancer cells.

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Section: Methodsmentioning

confidence: 99%

A Phosphatidic Acid (PA) conveyor system of continuous intracellular transport from cell membrane to nucleus maintains EGF receptor homeostasis

et al. 2016

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“…Taking advantage of the (T) 14 linker, the sense oligo was immobilized inititially to an N-oxysuccinimide ester-coated plate at a concentration of 200 pmol in a 100 μl volume per each well in oligonucleotide binding buffer (50 mM Na 3 PO 4 , pH 8.5, 1 mM EDTA) and washed extensively. The antisense oligo was added and a dsDNA was formed as in 37 . Plate-bound DNA was incubated with 30 ng/well PPARα for 20 min at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

Phosphatidic Acid (PA) can Displace PPARα/LXRα Binding to The EGFR Promoter Causing its Transrepression in Luminal Cancer Cells

Mahankali

Farkaly

Bedi

et al. 2015

Sci Rep

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The expression of the epidermal growth factor receptor (EGFR) is highly regulated in normal cells, whereas some cancer cells have high constitutive levels. Understanding naturally-occurring ways of downregulating EGFR in cancer cells was investigated. Phosphatidic acid (PA) or Nuclear Receptors (NR) PPARα/RXRα/LXRα, enhance EGFR expression, mediated by the promoter region -856(A) to -226(T). Unexpectedly, the combination of NRs and PA caused repression. PA induces a conformational change in the nuclear receptor PPARα (increase of alpha-helices at the expense of decreasing beta-sheets), as evidenced by circular dichroism. This represses the naturally-enhancing capability of PPARα on EGFR transcription. PPARα-overexpressing cells in the presence of PA > 300 nM or the enzyme that produces it, phospholipase D (PLD), downregulate EGFR expression. The reasons are two-fold. First, PA displaces PPARα binding to the EGFR promoter at those concentrations. Second, NR heterodimer-dependent promoter activity is weakened in the presence of PA in vivo. Since other genes considered (β-catenin, cyclin D3, PLD2 and ACOX-1) are also downregulated with a PA + PPARα combination, the transrepression appears to be a global phenomenon. Lastly, the reported effect is greater in MCF-7 than in MDA-MB-231 breast cancer cells, which could provide a novel basis for regulating excessive expression of EGFR in luminal cancer cells.

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“…No matter which kit is used, the optimal detection of TF activity ranges between 0.5 and 100 μg of nuclear extract. Wang et al [ 20 ] showed that the detection range was between 0.625 and 10 μg of nuclear extract. In our study, the sensitivity of TF activity was quantified as about 0.05–2 μg of nuclear extract.…”

Section: Discussionmentioning

confidence: 99%

A New Microsphere-Based Immunoassay for Measuring the Activity of Transcription Factors

Lin¹,

Lai²,

Tsai

et al. 2010

Biol Proced Online

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There are several traditional and well-developed methods for analyzing the activity of transcription factors, such as EMSA, enzyme-linked immunosorbent assay, and reporter gene activity assays. All of these methods have their own distinct disadvantages, but none can analyze the changes in transcription factors in the few cells that are cultured in the wells of 96-well titer plates. Thus, a new microsphere-based immunoassay to measure the activity of transcription factors (MIA-TF) was developed. In MIA-TF, NeutrAvidin-labeled microspheres were used as the solid phase to capture biotin-labeled double-strand DNA fragments which contain certain transcription factor binding elements. The activity of transcription factors was detected by immunoassay using a transcription factor-specific antibody to monitor the binding with the DNA probe. Next, analysis was performed by flow cytometry. The targets hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-κB) were applied and detected in this MIA-TF method; the results that we obtained demonstrated that this method could be used to monitor the changes of NF-κB or HIF within 50 or 100 ng of nuclear extract. Furthermore, MIA-TF could detect the changes in NF-κB or HIF in cells that were cultured in wells of a 96-well plate without purification of the nuclear protein, an important consideration for applying this method to high-throughput assays in the future. The development of MIA-TF would support further progress in clinical analysis and drug screening systems. Overall, MIA-TF is a method with high potential to detect the activity of transcription factors.

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Exonuclease-mediated ELISA-like assay for detecting DNA-binding activity of transcription factors: measurement of activated NF-κB

Cited by 10 publications

References 56 publications

A Phosphatidic Acid (PA) conveyor system of continuous intracellular transport from cell membrane to nucleus maintains EGF receptor homeostasis

A Phosphatidic Acid (PA) conveyor system of continuous intracellular transport from cell membrane to nucleus maintains EGF receptor homeostasis

Phosphatidic Acid (PA) can Displace PPARα/LXRα Binding to The EGFR Promoter Causing its Transrepression in Luminal Cancer Cells

A New Microsphere-Based Immunoassay for Measuring the Activity of Transcription Factors

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