A method for the quantitative analysis of stimulation-induced nuclear translocation of the p65 subunit of NF-κB from patient-derived dermal fibroblasts.
Developmental and immune-mediated disease has been linked to genetic mutation of key signaling components involved in NF-κB activation that leads to impaired activation or regulation of the canonical IKK complex. We identify patients with suspected or known defects of the NF-κB signaling pathway through clinical phenotyping and genetic sequencing. To help understand how mutations cause disease, we quantitate the kinetics and dose-response of NF-κB activation signaling events in their cells. Following activation of the canonical IKK complex, phosphorylation of the inhibitor of NF-κB proteins (IκB) leads to their degradation and the subsequent translocation of NF-κB family members from the cell cytoplasm to the nucleus. Here, we provide a method to obtain patient-derived dermal fibroblasts and quantitatively assess the integrity of the signal transduction pathway from receptor activation to nuclear p65 translocation.
Immunodeficiency and Inflammation Unit, Arthritis and Rheumatism Branch, NIAMS, National Institutes of Health, Bethesda, MD, 20892, USA.
Alex W
AW
Wessel
Eric P
EP
Hanson
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D001706:Biopsy; D018929:Cell Culture Techniques; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D012867:Skin / Q000166:cytology*; D051996:Transcription Factor RelA / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha