RNAi-Based Screening for the Identification of Specific Substrate-Deubiquitinase Pairs.

Ubiquitination signals are regulated in time and space due to the coordinated action of E3s and DUBs, which enables the precise control of cellular function and homeostasis. Mutations in all types of ubiquitin-proteasome system (UPS) components are related to pathological conditions. The identification of E3/DUBs' ubiquitinated substrates can provide a clearer view of the molecular mechanisms underlying those diseases. However, the analysis of ubiquitinated proteins is not trivial. Here, we propose a protocol to identify DUB/substrate pairs, by combining DUB silencing, specific pull-down of the substrate, and image analysis of its ubiquitinated fraction.