Immunoblot analysis of linear polyubiquitination of NEMO

Stimulation with inflammatory cytokines such as TNF-α and IL-1 activates the canonical NF-κB pathway through the activation of the IKK complex. The mechanism underlying IKK activation has been extensively studied and the involvement of the ubiquitin system has been well documented. We have recently reported that a novel ubiquitin ligase complex, LUBAC is involved in the activation of the IKK complex. LUBAC consists of one catalytic subunit, HOIP and two accessory molecules, HOIL-1L and SHARPIN and activates the IKK complex by conjugating the linear polyubiquitin chains to NEMO (IKKγ), the regulatory subunit of IKK complex. In this chapter, we describe the protocol for the detection of the linear polyubiquitination of NEMO by the immunoblotting using anti-linear ubiquitin antibody.