Additional parameters determined in data analysis were latency to the open arm, average speed, as well as percentage of test time and distance spent in the open and closed arms. Open field The OFT was utilized to examine locomotor activity as well as anxious behavior. A 16-beam animal activity monitor was used to divide the Plexiglass arena (40 cm L × 40 cm W × 30 cm H) into center and periphery. Fusion software (AccuScan Instruments Incorporated, Columbus, OH) analyzed various parameters based on

recorded activity, including total distance, Inhibitors,research,lifescience,medical entries, rest time, movement time, and latency to center and periphery. The movement time is the time spent by the animal moving, rather than freezing, in either the center or periphery. At the beginning of each test, every animal was introduced to the same corner (left back corner) of the arena and was allowed to explore the Inhibitors,research,lifescience,medical arena freely for 5 min. Animals were tested twice on consecutive days on the OFT to examine habituation. Sample collection and western blotting Two days after the last test, animals were deeply anesthetized with isoflurane inhalation and rapidly decapitated. The uteri from

the animals were removed and collected in preweighed tubes containing distilled water; wet weight and images of the whole dissected uteri were recorded. Inhibitors,research,lifescience,medical Dorsal and ventral hippocampus were Inhibitors,research,lifescience,medical dissected rapidly and homogenized in RIPA lysis buffer (Boston Bioproducts, Ashland, MA) containing both protease and phosphatase inhibitors (Sigma Aldrich, St. Louis, MO). Protein concentrations were determined by the Lowry assay (Bio-Rad, Hercules, CA). Total protein of 20 μg was separated with sodiumdodecyl Inhibitors,research,lifescience,medical sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using either

10% (for ERK and GAPDH) or 7.5% (for ERα and α-tubulin) acrylamide gels and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). Membranes were blocked for 1 h at room temperature with 5% bovine serum Selleck MS275 albumin (BSA) in Tris-buffered saline with 0.1% Tween 20 (TTBS) followed by incubation with mild agitation with the following primary antibodies diluted in blocking buffer: antiphospho-p44/42 mitogen-activated protein kinase Thr202/Tyr204 (1:2500; #4377, Cell Signaling Technology, MA) for 1 h at room temperature, antiphospho-S118 ERα (1:5000; sc-12915-R, Santa Oxygenase Cruz Biotechnology, CA) overnight at 4 C, anti-ERα (H-184) (1:1000; sc-7207, Santa Cruz Biotechnology, CA) overnight at 4 C, and anti-α tubulin (1:10,000; 1878-1, Epitomics, CA) for 1 h at room temperature. Following primary incubation, blots were washed with TTBS and incubated with anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (1:20,000; Cell Signaling Technology, MA) in 5% BSA/TTBS.