Figure 2. Stabilization of p53 upon Mdmx^F488A expression is post-translational.

(A) Following induction of Mdmx^WT or Mdmx^F488A, cycloheximide was added for the indicated times prior to blotting for p53 and Mdm2. Band intensities were calculated using the LiCor/Odyssey image analysis system, and plotted as log₂ values. (B) Mdm2 mRNA was analyzed by qPCR following addition of Dox, or the damaging agent NCS (300 ng/ml) as a positive control. (C) Cells were transfected with Histidine-tagged ubiquitin and doxycycline was added to induce Mdmx^WT or Mdmx^F488A. After 24h, cells were lyzed and ubiquitylated proteins were pulled down using Ni²⁺-agarose beads. Following SDS-PAGE, p53 was detected using DO-1. Numbers above each lane represent the ratio of ubiquitylated p53 species to the total amount of p53 in the lysate.