Selective monitoring of ubiquitin signals with genetically encoded ubiquitin chain-specific sensors.

Despite intensive research, there is a distinct lack of methodology for visualizing endogenous ubiquitination in living cells. In this protocol, we describe how unique properties of ubiquitin (Ub)-binding domains (UBDs) can be used to selectively detect, visualize and inhibit Ub-dependent processes in mammalian cells. The procedure deals with designing and validating the binding selectivity of GFP-tagged K63- and linear-linked sensors (TAB2 NZF and NEMO UBAN, respectively) in vitro. We describe how these moieties can be used to inhibit tumor necrosis factor (TNF)-mediated NF-κB signaling and to detect ubiquitinated cytosolic Salmonella in living cells, emphasizing a more flexible use compared with chain-specific antibodies. These chain-specific sensors can be used to detect Ub-like or autophagy-related modifiers and, in combination with mass spectrometry, to identify new Ub targets. These Ub (-like) sensors can be designed, constructed and tested in ~2-3 weeks.