Acetylation of p53 profound attenuation of p53 acetylation by 17b2E2 (E2), as compared to placebo-treated (Pla) controls at 24 h following cerebral ischemia (Fig. 1C). It should be mentioned that the regulatory effect of 17b2E2 on acetylation of p53 at Lys373 was only observed in animals following elevation by cerebral ischemia, as we observed no regulation by 17b2E2 of basal Acetyl p53- Lysine373 levels in non-ischemic sham controls (Figure S1A). Estrogen attenuates p53 acetylation at Lysine382 shortly after ischemic reperfusion Previous studies have shown that p53 can also be acetylated on Lysine 382, and that this increases stability of p53 [41]. We therefore examined whether Acetyl p53-Lysine382 levels increase in the hippocampal CA1 region at 3 h after GCI (the time when Acetyl p53-Lysine373 displayed peak levels) and determined whether 17b2E2 similarly attenuates acetylation of p53 at the Lys382 site. The results of the study revealed a marked increase in Acetyl p53-Lysine382 levels in the hippocampal CA1 region of placebo (Pla)-treated animals at 3 h after GCI as compared to sham controls. In addition, 17b2E2 significantly attenuated the elevation of Acetyl p53-Lysine382 levels (Figure S1B). Note that total p53 levels remained unchanged in all treatment groups examined, and that 17b2E2 had no effect on basal Acetyl p53- Lysine382 levels in non-ischemic sham animals. 17b-Estradiol Down-Regulates BH3 Family Members Puma and Noxa Following Ischemia We next examined the expression of the downstream p53 transcriptional target proteins, Puma and Noxa. In addition, we performed double immunohistochemistry on coronal hippocampal CA1 sections from the 24 h time-point for the neuronal marker, NeuN, (red) and Puma (green) to determine whether Puma exhibits neuronal localization and is regulated by 17b2E2. As shown in Fig. 2A, representative images revealed Puma predominantly localized in pyramidal neurons of the hippocampus CA1 region in all treatment groups studied. Of significant interest, the results revealed a robust elevation of Puma immunoreactive staining in placebo (Pla) animals following GCI as compared to sham controls, Figure 2. 17b-Estradiol attenuates downstream pro-apoptotic BH3 family members, Puma and Noxa, in the CA1 following ischemia. (A) Hippocampal CA1 sections were double stained for NeuN neuronal marker (red) and Puma (green) at 24 h following global cerebral ischemia. Merged images depict Puma neuronal localization in the CA1 region of the hippocampus. Magnification 40X. (B) Western blot using anti-Puma antibody shows Puma protein expression at 24 h following ischemic reperfusion. Actin bands represent loading control. Data is expressed as fold differences in comparison to sham from four to five animals. *p,0.05 vs. Sham, #p,0.05 vs. Pla; Pla = Placebo, E2 = 17b2E2. (C) Effect of 17b2E2 treatment on Noxa protein expression at 24 h reperfusion timepoint following cerebral ischemia. Representative photomicrographs of DAB staining obtained at a magnification of 10X (left panels). Semi-quantitative analysis of Noxa staining expressed as the number of Noxa-positive cells per 250 mm area obtained from 4–5 hippocampal sections per animal (right panels). *p,0.05 vs. Sham, #p,0.05 vs. Pla. n = 4–5 animals per treatment group. doi:10.1371/journal.pone.0027039.g002 PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e27039