Acetylation of p53 and 17b2E2 (E2) treatment strongly attenuated this elevation. Figure 2B shows Western blot analysis of total Puma protein levels in hippocampal CA1 region samples collected at 24 h after GCI. As shown in Fig. 2B, similar to the immunostaining results, Western blot analysis revealed that Puma levels are significantly elevated in placebo (Pla) animals after GCI as compared to sham controls, and that 17b2E2 (E2) treatment significantly attenuated the elevation of Puma. In Figure 2C, we examined an additional p53-induced BH3 family pro-apoptotic protein, Noxa, via DAB immunostaining. As shown in Fig. 2C (left panel), representative photomicro- graphs of Noxa immunostaining reveal a similar pattern to that of Puma up-regulation in ischemic animals, and 17b2E2 treatment markedly attenuating the ischemia-induced up-regulation of Noxa. Semi-quantitative analysis of the Noxa staining results through counting of the number of Noxa-positive cells in the hippocampal CA1 sections confirmed an elevation of Noxa-positive cells in the placebo (Pla) group as compared to the sham controls, and a strong attenuation of the number of Noxa-positive cells by 17b2E2 (E2) (Fig. 2C, right panel). 17b2E2 Enhances Both the Interaction of the Ubiquitin Ligase, Mdm2 with p53 and the Ubiquitination of p53 Following GCI It has been reported that deacetylation of p53 at Lysine373 decreases the stability of p53 and leads to its ubiquitination and degradation [42,43]. The E3 ubiquitin ligase, Mdm2, has been implicated to be an important factor responsible for ubiquitination of p53 [44,45]. We thus examined whether Mdm2 interaction with p53 and ubiquitination of p53 in the hippocampal CA1 region is modulated by 17b2E2 following GCI. We also examined p53 mRNA levels to rule out that p53 changes after GCI or 17b2E2 treatment were due to changes in p53 gene expression. Figure 3A illustrates the results of examination of p53 gene expression in the Figure 3. 17b-Estradiol enhances p53 interaction with Mdm2 and p53 ubiquitination. (A) P53 mRNA levels remain unchanged in the hippocampus CA1 at 6 h and 24 h following cerebral ischemia as measured by real-time RT-PCR. (B) 17b2E2 increases p53-ubiquitin interactions and decreases p53 levels at 24 h after GCI reperfusion. *p,0.05 vs. Pla. (C) 17b2E2 increases p53-Mdm2 binding two fold as compared to placebo-treated control levels at 6 h after cerebral ischemic injury. P53 protein expression level serves as Co-IP control. *p,0.05 vs. Pla. Pla = Placebo, E2 = 17b2E2.. n = 4–5 animals per treatment group. doi:10.1371/journal.pone.0027039.g003 PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e27039