Figure 2. Otub1 suppresses MDM2-mediated MDMX ubiquitination in cells and in vitro.

(A) (B) wt Otub1 and its C91S mutant, but not the D88A mutant, suppress MDM2-mediated p53 ubiquitination in cells. H1299 cells transfected with different combination of plasmids encoding His-Ub, myc-MDMX, HA-MDM2 in the absence or presence of Flag-Otub1, Flag-USP7 (A), or Flag-Otub1 (wt, C91S, or D88A mutant) (B) were subjected to Ni²⁺-NTA pulldown under denaturing conditions, followed by IB with anti-MDMX to detect the ubiquitinated species of MDMX (top panels). The protein expression is shown in bottom panels. (C) wt Otub1 and its C91S mutant, but not the D88A mutant, suppress MDM2-mediated MDMX ubiquitination in vitro. The in vitro ubiquitination reactions were performed in a reaction mixture containing recombinant Ub, E1, E2 (UbcH5), His-MDMX, GST-MDM2 in the absence or presence of His-Otub1 or its mutants (C91S or D88A), followed by IB with anti-MDMX to detect MDM2-mediated MDMX ubiquitination in vitro.