Figure 2

Proof-of-principle of the ULP method.

(a–c) HEK293 cells co-transfected with (a) Rnf8, (b) Traf6 or (c) Chfr ULP assay vectors as indicated (n = 3). Firefly/Renilla ratio serves as normalization control for transfection efficiency. The background level of luciferase is measured in the presence of empty vector controls and used for data normalization. E3 ligase mutants are used to demonstrate assay dependence on the catalytic activity of E3s. (d–f) U-2 OS cells co-transfected with (a) Rnf8, (b) Traf6 and (c) Chfr ULP assay vectors were treated with varying concentrations of Ubc13 inhibitor NSC697923 for two hours before cell lysis to determine the ULP assay activation. Data normalized to DMSO treated controls (n = 3).