ArticleJournal of immunological methods2015
Simultaneous assessment of NF-κB/p65 phosphorylation and nuclear localization using imaging flow cytometry.
Article in Journal of immunological methods, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 49 papers.
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Who cites it
49 citing papers in PubMed, 84 citations in OpenAlex.
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- Induction of cell death in ovarian cancer cells by doxorubicin and oncolytic vaccinia virus is associated with CREB3L1 activation.Molecular therapy oncolytics · 2021Article
- Therapeutic Benefit of Galectin-1: Beyond Membrane Repair, a Multifaceted Approach to LGMD2B.Cells · 2021Article
- Curcuma oil ameliorates benign prostatic hyperplasia through suppression of the nuclear factor-kappa B signaling pathway in rats.Journal of ethnopharmacology · 2021Article
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
Abstract
Aberrant activity of Nuclear Factor-kappaB (NF-κB) is associated with many diseases and is therapeutically targeted. Post-translational modifications, particularly phosphorylation of the RELA/p65 sub-unit, are essential for cytoplasmic to nuclear localization of NF-κB/p65 and initiation of transcription of downstream target genes. Immunoblot and phospho-flow cytometry have been used to study the relationship between phosphorylation motifs and NF-κB activation and microscopic analysis of nuclear localization of p65 is also used as a parameter for activation. The labor intensive nature of these approaches commonly limits the number of sampling points or replicates. Recent insights into the relationship between p65 phosphorylation motifs and their nuclear localization indicate that these parameters have different significances and should not be used interchangeably. In this study, we demonstrate feasibility and reproducibility of studying the relationship between p65 phosphorylation and nuclear translocation using imaging flow cytometry (IFC). TNFα- or PMA/Ionomycin-induced phosphorylation of p65 at serine 529 in cell line models and healthy donor lymphocytes served as the experimental model. IFC analysis demonstrated that expression of phosphorylated serine 529 (P-p65(s529)) increased rapidly following stimulation and that nuclear localization of P-p65(s529) followed the nuclear localization pattern of total p65. However, in the presence of tacrolimus, P-p65(s529) expression was inhibited without affecting nuclear localization of total p65. The data demonstrate the application of IFC to simultaneously assess phosphorylation of p65 and its cellular localization and the results obtained by this analysis corroborate current insights regarding the specific effect of tacrolimus on serine 529 phosphorylation.
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