Fig 2: Nuclear sequestration reduces MDMX activity to suppress p53 protein expression. A MDM2487/487 MEF cells were treated with empty vector (EV), MDMX or NLS-tagged MDMX lentiviral constructs for 48 h prior to immunostaining using an anti-MDMX antibody, and DAPI nuclear staining. B MDM2487/487 MEFs were treated with empty vector (EV), MDMX, or NLS-tagged MDMX lentiviral constructs for 48 h prior to lysis and immunoblotting

Fig 3: DNA damage inhibits MDMX suppression of p53 protein expression. MDM2487/487 MEFs were treated with empty vector (EV) or MDMX-expression lentiviral constructs for 48 h prior to lysis. For DNA damage conditions, MEF cells were treated with 1μg/mL doxorubicin for four hours, or 10 Gy ionizing irradiation (IR) for six hours prior to lysis and immunoblotting

Fig 4: MDMX suppresses p53 protein levels in MDM2487/487 MEFs. A WT (MDM2+/+) and MDM2487/487 MEFs were lysed and subject to immunoblotting for the indicated proteins. B WT and MDM2487/487 MEFs were treated with nonspecific (NS) siRNA, or MDMX-targeting siRNA for 32 h prior to lysis and immunoblotting. C WT and MDM2487/487 MEFs were transduced with nonspecific (NS), or MDMX-targeting shRNA lentiviral constructs for 48 h in the presence of 1μg/mL puromycin prior to lysis and immunoblotting

Fig 5: MDM2-MDMX binding is essential for MDMX-mediated suppression of p53 protein levels. A MDM2487/487 MEF cells were treated with increasing volumes of MDMX-expressing lentiviral constructs for 48 h prior to lysis and immunoblotting. B, C HEK293 cells were transfected with GFP vector and FLAG-MDM2 along with Myc-tagged MDMX constructs for 24 h prior to lysis and immunoprecipitation (IP). 1 mg of lysate was subject to IP using anti-FLAG beads prior to immunoblotting using an anti-Myc antibody. D MDM2487/487 MEFs were treated with empty vector (EV), lentiviral constructs expressing full-length MDMX, acidic binding domain deletion (ΔAD), or RING domain deletion (ΔRING) MDMX for 48 h prior to lysis and immunoblotting