Figure 5. Mdm2 inhibits binding of L26 to p53 mRNA.

MCF7 cells (3×10⁶ cells/10cm dish) were co-transfected with expression plasmids encoding GFP (4μg/dish); lanes 1,5) or GFP-L26 (4μg/dish); lanes 2–4, 6–8), without or with increasing amounts of human Mdm2 (4μg or 8μg/dish) expression plasmid. The total amount of DNA was kept at 12μg by the inclusion of plain pCMV-Neo-Bam vector. Forty-eight hours post-transfection, cultures were exposed to ionizing radiation (IR; 10Gy) as indicated and harvested 30 minutes post-IR. Lysates were immunoprecipitated with anti-GFP antibodies and the amount of coprecipitated p53 mRNA was assessed by IP-RT-PCR, employing primers that map within the 5′-UTR of p53 mRNA and span an intron within the genomic human p53 DNA. PCR products were visualized by ethidium bromide staining (top panel). Protein input for each IP reaction was resolved by SDS-PAGE and assessed by Western blot analysis with the indicated antibodies (bottom panels).