Figure 1

Design of the ULP method.

CheckMateTM/Flexi® mammalian two-hybrid vectors modified as indicated in Materials and Methods to express a fusion of the Gal4 DNA-binding domain (DBD, purple) to an E3 ubiquitin ligase (orange) and a fusion of the VP16 activation domain (ACT, red) to TUBEs (green) are co-transfected into mammalian cells along with the firefly luciferase reporter vector, which contains five Gal4 binding sites upstream of the minimal TATA box. Binding of DBD to the Gal4 cassette at the luciferase promoter results in the recruitment of the E3 ubiquitin ligase to the reporter vector (step 1). The E3 ligase autoubiquitylation activity results in decoration of poly-ubiquitin chains (blue ovals) on the ligase surface, creating binding sites for TUBEs (step 2). This subsequently leads to the recruitment of ACT to the minimal TATA box (step 3), thereby linking the E3 catalytic activity under study to the expression of the firefly luciferase reporter, the activity of which is detected following cell lysis (step 4).