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.
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We thank A. Bremm, D. McEwan and J. Lopez for critical reading of the manuscript. This work was supported by the Cluster of Excellence 'Macromolecular Complexes' of the Goethe University, Frankfurt am Main (EXC115); LOEWE Oncology Signaling Network and LOEWE Center for Cell and Gene Therapy, Frankfurt am Main; and a European Research Council Advance Grant (I.D).
The authors declare no competing financial interests.
Schematic representation of potential sensors for Ub, UBLs and autophagy-like modifiers (PDF 598 kb)
Schematic representation of mammalian Ub sensors used in this protocol (PDF 582 kb)
Stably expressed GFP NEMO UBAN co localizes with Ub chain specific antibodies on Infected Salmonella (PDF 620 kb)
Expression of TAB2 NZF E685A does not affect TNF induced NF-κB signalling (PDF 616 kb)
Recruitment of linear Ub chain-specific NEMO UBAN to cytosolic Salmonella in living cells (AVI 1951 kb)
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van Wijk, S., Fiškin, E. & Dikic, I. Selective monitoring of ubiquitin signals with genetically encoded ubiquitin chain–specific sensors. Nat Protoc 8, 1449–1458 (2013). https://doi.org/10.1038/nprot.2013.089
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