Western blotting

To quantify CREB phosphorylation and immunocontent, western blotting analysis was performed as previously described [47, 54, 55]. Animals were euthanized by decapitation; brains were excised from the skull; and hippocampi and prefrontal cortex were dissected into cold saline solution, placed in liquid nitrogen, and then stored at −80 °C until use. Briefly, samples were mechanically homogenized in 300 μl of 50 mM Tris (pH 7.0), 1 mM EDTA, 100 mM NaF, 0.1 mM PMSF, 2 mM Na₃VO₄, 1% Triton X-100, 10% glycerol, and Amresco protease inhibitor cocktail catalog number M222 (working concentration 0.5 mM AEBSF, 0.3 μM aprotinin, 10 μM bestatin, 10 μM E-64, 10 μM leupeptin, 50 μM EDTA). Lysates were centrifuged (10,000g for 10 min, at 4 °C) in order to eliminate cellular debris. The supernatants were diluted 1/1 (v/v) in 100 mM Tris (pH 6.8), 4 mM EDTA, and 8% SDS, followed by boiling for 5 min. Thereafter, samples were diluted in 40% glycerol, 100 mM Tris, and bromophenol blue (pH 6.8) in the ratio of 25:100 (v/v), and β-mercaptoethanol at a final concentration of 8% was added to each sample. Protein content was estimated by the method described by Peterson [56]. The samples (60 μg of total protein/track) were electrophoresed in 10% SDS-PAGE minigels and transferred to nitrocellulose membranes using a semi-dry blotting apparatus (1.2 mA/cm²; 1.5 h). To verify transfer efficiency process, membranes were stained with Ponceau stain. The membranes were blocked with 5% bovine serum albumin (BSA) in TBS (10 mM Tris, 150 mM NaCl, pH 7.5). The total and phosphorylated forms of CREB and β-actin were detected after overnight incubation with specific antibodies diluted in TBS-T containing 2% BSA. The primary rabbit antibodies were diluted 1:1000 for the phosphorylated (Ser133) and total forms of CREB (Cell Signaling) and 1:2000 for mouse anti-β-actin (Santa Cruz Biotechnology). Membranes were incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated antirabbit or antimouse antibody (1:5000, Millipore) for protein detection. The reactions were developed by chemiluminescence substrate (LumiGLO). After blocking and incubation steps, membranes were washed three times (5 min) with TBS-T (10 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.5). The bands were quantified using the Scion Image® software. CREB phosphorylation was determined as a ratio of optical density (OD) of phosphorylated band/OD of total band, and the expression of CREB was determined as a ratio of OD CREB band/OD of β-actin band.