Fig. 1

(a) Schematic representation of MDM2 and the splice variants MDM2-A, -B and -C. MDM2 consists of 491 amino acids. Localization of the p53, pRb and MDM2/4 binding sites, NLS, NES, NoLS, the acidic domain, the Zn-finger domain and the RING-finger domain are indicated, as well as the exon distribution. (b) Validation of protein expression. MCF-7 breast cancer cells transfected with pCMV-GFP control (GFP-control), MDM2-A (75 kDa), -B (48 kDa) and -C (85 kDa) analyzed 24 h post transfection. GAPDH was used as loading control. Primary antibodies were Anti-MDM2 (N-20) Sc-813 (Santa Cruz) and GAPDH (SantaCruz). (c) Protein stability of the expressed splice variants. MCF-7 cells transfected with MDM2-FL, MDM2-A, -B and -C at 0, 1, 2 and 4 h post cycloheximide treatment, respectively. In addition to cycloheximide treatment, left panel shows cells without doxorubicin treatment, right panel shows cells treated with 1 μM doxorubicin for 24 h. Primary antibodies Anti-MDM2 (N-20) Sc-813 (Santa Cruz) and GAPDH (SantaCruz). Histograms under immunoblots represent averages of triplicate experiments and show levels of the MDM2-variants relative to GAPDH-levels for each sample