Nuclei aliquots were resuspended in 200 mL of a 0.2 M solution of boric acid, pH 9.0, and heated for 1 h at 75uC for epitope retrieval. Subsequently, nuclei were incubated for 24 h at 4uC in primary antibodies (1:500 for polyclonal anti-Ki-67, abcam, ab15580) and in CY2-conjugated goat anti-rabbit IgG secondary antibody (1:100 in PBS; Jackson Immuno Reasearch Labs, Inc.) for 2 h. An aliquot of the nuclear suspensions (2.5 ul) was stained with propidium iodide (PI), a fluorescent molecule that stoichio- metrically binds to DNA by intercalating between the bases with no sequence preference, and checked under fluorescent micro- scope to verify the immunolabeling quality. The remaining cell suspension was diluted to 500 mL for flow cytometry assay using a Beckman FC 500 System with CXP Software. To avoid counting bias, we registered the presence or absence of Ki-67 immunore- activity for all of the PI-stained nuclei samples until 10,000 PI- stained nuclei were examined. Nuclear density was estimated by counting the PI positive particles. Extraction of leukocytes protein from whole blood To translate the concept that ERb2 expression predicts the effectiveness of ET into a clinically useful guideline, we measured ERb2 expression in circulating white blood cells (WBC). Ammonium chloride solution (0.17 M at room temperature) was added to each blood sample in a volume 1:9 to lyse red blood cells and the samples incubated for 5 minutes in a rotator. The red blood cells would not be completely lysed if the solution was cold and the white blood cells began to lyse if the incubation exceeded 5 minutes. The suspension of WBC and lysed RBCs was centrifuged for 5 minutes at 2000 RPM. The supernatant was discarded and the pellet washed in ice old PBS (in a volume of 10 times of that of starting blood sample) [34]. The white blood cells were collected by 5 min centrifugation at 2000 RPM and lysed in 0.2 ml of ice cold fresh RIPA buffer. After suspension by gentle pipetting, the lysates were set on ice for 15 minutes to increase the total yield of soluble protein. Samples were then sonicated on a 70% setting for 30 sec to shear the DNA, incubated 30– 60 minutes on ice, centrifuged on a bench-top microcentrifuge at 12,0006g for 15 minutes at 4uC, and the supernatant collected into a new tube. The protein concentration was determined by the BCA method (Pierce Biotechnology, Rockford, IL). Western blot Two specific antibodies were used to detect ERb and ERb2 protein. The H-150 antibody recognizes the N-terminal epitope of rat ERb (Santa Cruz) and the ERb2 antibody (gift from Dr. Robert Handa) recognizes the unique 18 aa residues in ERb2 and does not cross-react with either ERb, ERa, or other ER isoforms [25]. Thus a protein band on PVDF membrane that is positive for both the H-150 and Handa’ antibodies reduced the possibility of a false positive for ERb2. Antibodies for b-actin, a loading control for cytosol protein, and histone H3, a nuclear protein loading control, were used as internal controls. The relative quantity of ERb and ERb2 were normalized by internal controls. The data were collected with a Bio-Rad Molecular Imager ChemiDoc XRS+ system and the optical density was analyzed with the Bio- Rad image lab software. Forced Swim Test Rats were placed in a cylindrical container (40 cm deep, 27 cm in diameter) filled with 30 cm of 30uC water, the 10-min test was videoed, and the amount of time the rats spent swimming or immobile in 10-min was analyzed. Swimming was defined as movement of the forelimbs and hindlimbs without the front paws breaking the surface of the water. Immobility was recorded when ERb2 Determines the Therapeutic Window of ET there was an absence of any movement other than that necessary to keep the head and nose above the water (when rats were floating in a vertical position) [35,36]. Before testing, all rats were coded and codes were not broken until the video analyses had been completed. Statistics Statistically analysis was by a Two-way ANOVA followed by a Neuman-Keuls test. In all analyses, differences were considered significant at probability (p) values less than 0.05. Results Removal of ovaries increases the expression of a dominant negative ERb splice variant, rERb2 The role of ovarian hormones on formation of the dominant negative ERb splice variant, ERb2 (an intron retention isoform), was first tested in hippocampus of 6 month old reproductive female rats. The ERb specific antibody (h-150, Santa Cruz) detected two proteins at molecular masses of 54 kDa and 57 kDa (Figure 2A). The 57 kDa larger band is consistent with ERb2 which has an additional 18 amino acid in the ligand binding domain of ERb. The ratio of the 57/54 kDa proteins in na¨ıve rats is 4.7%, in sham rats is 3.6%, and in OVX rats is 112% (Figure 2B). To identify the 57 kDa protein band which is recognized by ERb antibody h-150, an antibody which specifically recognizes the 18 amino acid in ERb2, but does not cross-react with ERb or ERa [25] was used. The 57 kDa protein band was only recognized by the antibody against the unique 18 amino acids in ERb2 (Figure 2C). ERb2 expression was detectable in 6 month- old female OVX rats 15 days after OVX, but not in sham OVX rats (Figure 2C). Figure 2. Loss of ovarian hormones increases ERb2 expression. Each lane was loaded with 30 mg of protein from rat hippocampus (n = 6/group). A. Western blot of ERb in hippocampus of 6 month old rats that were naive, or sham and OVX for 14 days using h-150 ERb antibody (Santa Cruz). B. Optical density of the expressed 54 and 57 kDa ERb immunoreactive proteins using the h-150 ERb antibody. C. Western blot using the ERb2 antibody (gift from Dr. Handa) which recognizes the unique 57 kDa 18 amino acid sequence in rERb2that does not cross-react with either ERb or ERa. doi:10.1371/journal.pone.0033493.g002 PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e33493