Figure 2

Identification of TRABID DUB inhibitors. A,B) Effects of compounds on TRABID DUB activity. Compounds (30 μM in A) were incubated with Hexa-K63 ubiquitin and immunoprecipitated FLAG-TRABID for 3 hrs. Samples were then analyzed by Western under a non-reducing condition using an antiubiquitin antibody. The DUB activity was quantified in B by determining the optical density of hexa-K48 ubiquitin bands at 50 kD. The band without TRABID was taken as 100% and the one with TRABID and DMSO as 0%. The experiments were repeated three times in duplicates, and errors are less than 5%. C,D) Effects of compounds on A20 DUB activity. Hexa-K63 ubiquitin was incubated with compounds and immunoprecipitated FLAG-A20 and analyzed by Western under a nonreducing condition using an anti-ubiquitin antibody. Quantification of DUB activity in D was carried out as described in B. E) Chemical structures of NSC112200, NSC267309 and NSC60650. F) Molecular modeling of the binding of NSC112200 to TRABID OTU G) Molecular modeling of the binding of NSC267309 to TRABID OTU H) Molecular modeling of the binding of NSC60650 to TRABID OTU.