Fig. 6

Characterization of ubiquitination inhibitor selectivity. BAY 11-7082, CC0651, clomipramine, heclin, and PYR-41 were tested for selectivity in a multi-step UPS-CONA assay. a Effects of inhibitors on the Ube1-Ube2L3-E6AP cascade. Inhibition of Ub charging to Ube1 was measured as decrease in fluorescent ubiquitination signal on Ube1 prior to adding Ube2L3 to the reaction. Inhibition of the Ube1-Ube2L3 transfer was measured as a decrease of Ub signal from Ube1 after adding Ube2L3. Final effect on E6AP ubiquitination was measured as a decrease in ubiquitin conjugation levels to E6AP. Detected signal was normalized to DMSO controls and fitted where possible by non-linear regression to the four-parameter logistic model using GraFit 7. b Comparison of inhibition effects on Ube1 observed using UPS-CONA. Ube1 was immobilized on beads, preincubated with inhibitors, and subjected to UPS-CONA. On the left, dose-dependent inhibition of ubiquitin loading to immobilized Ube1 was observed for PYR-41, BAY 11-7082, and heclin. On the right, quantification of the non-thioester-bound ubiquitin fraction remaining after treatment with DTT revealed potentially different mechanisms of action. c Gel-based assay for BAY 11-7082 and heclin inhibition of Ube1. Ube1 was preincubated in the ubiquitination buffer with inhibitors at indicated concentrations prior to adding WT-ubiquitin and ATP. After 5 min at 30 °C, the reactions were stopped by adding SDS loading buffer. Samples were resolved by SDS-PAGE and revealed by Western blot with anti-ubiquitin and anti-Ube1 antibodies. d Ube1 activity (Ube1~Ub thioester ratio to total Ube1) was quantified using densitometric analysis (ImageJ). The chart shows corresponding inhibition curve generated in at least two independent experiments