. Author manuscript; available in PMC: 2021 Mar 2.

Published in final edited form as: Nat Struct Mol Biol. 2020 Jun 15;27(7):605–614. doi: 10.1038/s41594-020-0438-0

Table 3.

Validation methods for targeted protein degradation.

Steps	Purpose	Example Assays
Dimerization – in vitro	Ternary complex formation in vitro	TR-FRET^40,43, SPR/BLI¹¹⁵
Dimerization – cellular	Ternary complex formation in cells	NanoBit¹¹⁶ NanoBret¹¹⁶
Cellular engagement	Cellular permeability and cellular engagement of the E3 ligase	Degradation based engagement assay⁴³ NanoBret based Fluoro-ligand displacement¹¹⁷
Ubiquitination – in vitro	Verify ubiquitination	In-vitro ubiquitination assay
Ubiquitination – cellular	Ubiquitination quantification and type	Western blot, NanoBret, TUBES¹¹⁸
Ubiquitination – cellular	Identify ubiquitination sites	Proteomics
Cellular degradation – targeted approach	Verify degradation in cells	Western blot, GFP-fusion, mCherry reporter lines¹¹⁹ Endogenous CRISPR fusions (HiBit Tag¹¹⁶, Split GFP¹²⁰)
Cellular degradation – selectivity profiling by proteomics	Verify degradation selectivity in cells	Proteomics approaches^32,52, library based screens⁴⁰
UPS inhibitors	Inhibitors for Cullin-RING family of ligases^121,122	MLN4924 - a specific inhibitor of the NAE1/UBA3 Nedd8 activating enzyme CSN5i-3 – inhibitor of COP9 signalosome
	p97 inhibitor¹²³	CB-5083
	Ubiquitin E1 (UBA1) inhibitor	MLN7243
	Proteasome inhibitors¹²⁴	Bortezomib, Carfilzomib and MG132