Figure 2.

Example of p53 ubiquitination assay for selected compounds (refer Table 1). Western blots with anti-p53 antibody of SDS–PAGE separation of reaction mixtures after incubation of immobilised HDM2 with test compounds or diluent only (C) and p53, ubiquitin, and ubiquitin ligases (-E2 indicates reactions omitting E2 ligase). Prescreen at 50–250 μM for compounds with different activity levels (a) and titration of compound 15 (b). The positions of p53, mono-ubiquitinated (Ub-53), and poly-ubiquitinated p53 (Ub_n-p53) are indicated.