Supplementary Figure 2 from Proteasome Addiction Defined in Ewing Sarcoma Is Effectively Targeted by a Novel Class of 19S Proteasome Inhibitors

Panel upper left: CHP100 cells exposed 3 hours to different concentrations of b-AP15. Extracts were labeled with HA-Ub-VS and subjected to western blot. Filters probed with HA antibody. Note the lack of inhibition of any of the DUBs labeled by HA-Ub-VS. Panel upper right: CHP100 cells exposed 3 hours to 1 uM b-AP15 Extracts were labeled with Ub-VS and subjected to western blot. Filters probed with USP14, UCHL5 or UCHL1 antibodies. b-AP15 treated extracts demonstrate inhibition of USP14, weak inhibition of UCHL5, and no inhibition of UCHL1. Middle Panels: Purified 26S proteasomes (5 nM) were treated with indicate concentrations for b-AP15 and DUB activity was determined by cleavage of Ub-AMC. Note the reduction in 26S associated proteasome activity following treatment with b-AP15. Lower Panels: Recombinant UCHL1 or UCHL3 was treated with b-AP15 and DUB activity was monitored using Ub-AMC cleavage as above. No inhibition of either UCHL1 or UCHL3 was observed.