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Updated: Apr 16, 2026
Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates
Published on: May 10, 2022
Immunoblot analysis of linear polyubiquitination of NEMO
Yoshiteru Sasaki1, Hiroaki Fujita, Misa Nakai
1Department of Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan.
Insights
The LUBAC complex conjugates linear polyubiquitin chains to NEMO, activating the IKK complex. This study details a method to detect NEMO linear polyubiquitination using immunoblotting.
Area of Science:
- Molecular Biology
- Immunology
- Cell Signaling
Background:
- Inflammatory cytokines like TNF-α and IL-1 activate the NF-κB pathway via the IKK complex.
- The ubiquitin system plays a documented role in IKK complex activation.
- The novel LUBAC (Linear Ubiquitin Assembly CHain) complex, comprising HOIP, HOIL-1L, and SHARPIN, has been identified as crucial for IKK activation.
Purpose of the Study:
- To describe a protocol for detecting linear polyubiquitination of NEMO (NF-κB essential modulator).
- To provide a method for studying the role of LUBAC in NF-κB pathway activation.
Main Methods:
- Immunoblotting technique using a specific anti-linear ubiquitin antibody.
- Detection of linear polyubiquitin chains conjugated to NEMO (IKKγ).
Main Results:
- The study focuses on the methodology rather than presenting experimental results.
- The described protocol enables the visualization of linear ubiquitination on NEMO.
Conclusions:
- Linear polyubiquitination of NEMO by LUBAC is a key mechanism in IKK complex activation.
- The described immunoblotting protocol is essential for investigating LUBAC-mediated NF-κB signaling.
Abstract:
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.
