Immunoblot Analysis of the Regulation of TNF Receptor Family-Induced NF-κB Signaling by c-IAP Proteins.

Proper maintenance of organismal homeostasis, development, and immune defense requires precise regulation of survival and signaling pathways. Inhibitor of apoptosis (IAP) proteins are evolutionarily conserved regulators of cell death and immune signaling that impact numerous cellular processes. Although initially characterized as inhibitors of apoptosis, the ubiquitin ligase activity of IAP proteins is critical for modulating various signaling pathways (e.g., NF-κB, MAPK) and cell survival. Cellular IAP1 and 2 regulate the pro-survival canonical NF-κB pathway by ubiquitinating RIP1 and themselves thus enabling recruitment of kinase (IKK) and E3 ligase (LUBAC) complexes. On the other hand, c-IAP1 and c-IAP2 are negative regulators of noncanonical NF-κB signaling by promoting ubiquitination and consequent proteasomal degradation of the NF-κB-inducing kinase NIK. Here we describe the involvement of c-IAP1 and c-IAP2 in NF-κB signaling and provide detailed methodology for examining functional roles of c-IAPs in these pathways.