ArticleMolecular & cellular proteomics : MCP2011
Quantification of activated NF-kappaB/RelA complexes using ssDNA aptamer affinity-stable isotope dilution-selected reaction monitoring-mass spectrometry.
Article in Molecular & cellular proteomics : MCP, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 29 papers.
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Who cites it
29 citing papers in PubMed, 46 citations in OpenAlex.
- Engineered anchoring aptamers induce relocalization and functional inactivation of transcription factors.Science advances · 2026Article
- A Branched SELEX Approach Identifies RNA Aptamers That Bind Distinct HIV-1 Capsid Structural Components.ACS infectious diseases · 2024Article
- Evolution of proteomics technologies for understanding respiratory syncytial virus pathogenesis.Expert review of proteomics · 2021Article
- Gibberellin JRA-003: A Selective Inhibitor of Nuclear Translocation of IKKα.ACS medicinal chemistry letters · 2020Article
- Evaluation of Aptamers as Affinity Reagents for an Enhancement of SRM-Based Detection of Low-Abundance Proteins in Blood Plasma.Biomedicines · 2020Article
- Quantitative Proteomics of the Endothelial Secretome Identifies RC0497 as Diagnostic of Acute Rickettsial Spotted Fever Infections.The American journal of pathology · 2020Article
- Application of targeted mass spectrometry in bottom-up proteomics for systems biology research.Journal of proteomics · 2018Review
- Quantitative Assessment of the Effects of Trypsin Digestion Methods on Affinity Purification-Mass Spectrometry-based Protein-Protein Interaction Analysis.Journal of proteome research · 2017Article
- Systematic Analysis of Cell-Type Differences in the Epithelial Secretome Reveals Insights into the Pathogenesis of Respiratory Syncytial Virus-Induced Lower Respiratory Tract Infections.Journal of immunology (Baltimore, Md. : 1950) · 2017Article
- BRD4 Couples NF-κB/RelA with Airway Inflammation and the IRF-RIG-I Amplification Loop in Respiratory Syncytial Virus Infection.Journal of virology · 2017Article
- Coordinate activities of BRD4 and CDK9 in the transcriptional elongation complex are required for TGFβ-induced Nox4 expression and myofibroblast transdifferentiation.Cell death & disease · 2017Article
- BRD4 mediates NF-κB-dependent epithelial-mesenchymal transition and pulmonary fibrosis via transcriptional elongation.American journal of physiology. Lung cellular and molecular physiology · 2016Article
- Generation and characterization of a novel transgenic mouse harboring conditional nuclear factor-kappa B/RelA knockout alleles.BMC developmental biology · 2016Article
- Review
- Article
- Systematic Determination of Human Cyclin Dependent Kinase (CDK)-9 Interactome Identifies Novel Functions in RNA Splicing Mediated by the DEAD Box (DDX)-5/17 RNA Helicases.Molecular & cellular proteomics : MCP · 2015Article
- Anti-peptide monoclonal antibodies generated for immuno-multiple reaction monitoring-mass spectrometry assays have a high probability of supporting Western blot and ELISA.Molecular & cellular proteomics : MCP · 2015Article
- Inducible STAT3 NH2 terminal mono-ubiquitination promotes BRD4 complex formation to regulate apoptosis.Cellular signalling · 2014Article
- Modulation of gene expression regulated by the transcription factor NF-κB/RelA.The Journal of biological chemistry · 2014Article
- Article
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Authors and funding
7 authors at 1 institution in 1 country.
Funding
Abstract
Nuclear Factor-κB (NF-κB) is a family of inducible transcription factors regulated by stimulus-induced protein interactions. In the cytoplasm, the NF-κB member RelA transactivator is inactivated by binding inhibitory IκBs, whereas in its activated state, the serine-phosphorylated protein binds the p300 histone acetyltransferase. Here we describe the isolation of a ssDNA aptamer (termed P028F4) that binds to the activated (IκBα-dissociated) form of RelA with a K(D) of 6.4 × 10(-10), and its application in an enrichment-mass spectrometric quantification assay. ssDNA P028F4 competes with cognate duplex high affinity NF-κB binding sites for RelA binding in vitro, binds activated RelA in eukaryotic nuclei and reduces TNFα-stimulated endogenous NF-κB dependent gene expression. Incorporation of P028F4 as an affinity isolation step enriches for serine 536 phosphorylated and p300 coactivator complexed RelA, simultaneously depleting IκBα·RelA complexes. A stable isotope dilution (SID)-selected reaction monitoring (SRM)- mass spectrometry (MS) assay for RelA was developed that produced a linear response over 1,000 fold dilution range of input protein and had a 200 amol lower limit of quantification. This multiplex SID-SRM-MS RelA assay was used to quantify activated endogenous RelA in cytokine-stimulated eukaryotic cells isolated by single-step P028F4 enrichment. The aptamer-SID-SRM-MS assay quantified the fraction of activated RelA in subcellular extracts, detecting the presence of a cytoplasmic RelA reservoir unresponsive to TNFα stimulation. We conclude that aptamer-SID-SRM-MS is a versatile tool for quantification of activated NF-κB/RelA and its associated complexes in response to pathway activation.
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