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.