Figure 3. Mdm2 Ser394 phosphorylation is necessary for p53 stability and activity after DNA damage.

(A) Mice were either untreated or irradiated with 4Gy and spleens were harvested 4 hours later. Protein levels were then analyzed by western blot.

(B) Mice were either untreated or irradiated with 4Gy and spleens were harvested 4 hours later (n=3 per genotype). Relative expression levels of p53 target genes in irradiated spleen extracts were determined by qRT-PCR. Levels were normalized to untreated wild-type and are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. Asterisks indicate a significant difference: double asterisk Puma p=0.002, p21 p=0.01, Bax p=0.003, Noxa p=0.002; single asterisk Puma p=0.01, p21 p=0.001, Bax p=0.01, Noxa p=0.008.

(C) Mice were either untreated or irradiated with 4Gy and thymii were harvested at the indicated times. Protein levels were then analyzed by western blot.

(D) Mice were either untreated or irradiated with 4Gy and thymii were harvested 2 or 4 hours later (n=3 per genotype). Relative expression levels of p53 target genes were determined by qRT-PCR. Data are presented as the ratio of mRNA to Gapdh mRNA. Standard deviation indicated by error bars. Significant differences were seen between WT and A/A Puma at 2 (p=0.035) and 4 (p=0.002) hours and between WT and A/A p21 at 2 (p=0.0001) and 4 (p=0.0001) hours.

(E) Protein levels from whole thymus extracts from untreated mice were analyzed by western blot (n=3 per genotype). Quantified levels of p53 (p=0.83) and Mdm2 (p=0.11) relative to PI3K were normalized to wild-type.

(F) Mice were either untreated or irradiated with 4Gy and thymii were harvested at the indicated times. Protein levels were then analyzed by western blot. Quantified levels of p53 and Mdm2 relative to PI3K were normalized to untreated wild-type. See also Figure S3.