Analyze the SUMOylation of IKK γ/NEMO During Genotoxic Stress.

SUMOylation is an important posttranslational modification of substrate proteins that regulates their functions in a variety of cellular processes including epigenetic and transcriptional regulation of gene expression, genomic stability, DNA repair, subcellular translocation, and protein turnover. The critical roles of SUMOylation in regulating NF-κB signaling is exemplified by the findings that it regulates IκBα stability, transactivity of RelA and RelB, as well as initiating the export of nuclear DNA damage signal to cytoplasmic IKK complex through NEMO SUMOylation. Detection of SUMOylated protein is technically challenging due to only a small fraction of substrate proteins is SUMOylated and this process is also reversible by highly active SUMO-deconjugating enzymes. In this protocol, we outline a method for detecting SUMOylation of NEMO in mammalian cells treated by genotoxic agents.