Figure 3. S7 Inhibits MDM2 E3 Ligase Activity in a Manner that Involves MDMX.

(A) Purification of MDM2/MDMX complex. Increasing amounts (0.05, 0.1 and 0.2 µg) of Flag-MDM2, HA-MDMX or MDM2/MDMX complex purified from insect cells were resolved on SDS-PAGE gel and visualized by silver staining. Amounts of the complex used in subsequent experiments were adjusted so that total amounts of MDM2 protein were equivalent. (B) S7 inhibits MDM2 E3 activity only when it is present in an MDM2/MDMX complex. Ubiquitination reaction mixtures containing either 0.1 or 0.3 µg of MDM2 alone or the MDM2/MDMX complex with or without 0.5 µg S7 protein as indicated, along with ~300 pmol ubiquitin, UbcH5c and rabbit E1 were incubated at 37°C for 40 min. Reactions were terminated by addition of SDS-sample buffer and mixtures with equivalent amounts of MDM2 were resolved by SDS-PAGE. Ubiquitin conjugates were visualized by Western blotting with anti-MDM2 antibody. Migration of mono- (Ub₁) di- (Ub₂), and poly- (Ub_n) ubiquitinated MDM2 species are indicated at left. (C) S7 and MDMX increase MDM2 stabilization. U2OS cells were transfected with the indicated plasmids (1.2 µg each). Cells were harvested after 24 hours and cell lysates were subjected to IB with indicated antibodies. GFP construct was added for transfection and loading control.