Rabbit anti creb antibody

After the dorsal and ventral hippocampal slices were harvested, the DG regions were micro-dissected and stored at −80°C until assayed. Protein was extracted and the concentration of protein was determined using a bicinchoninic acid (BCA) protein assay kit (Pierce, IL, USA). Protein (40 μg) was separated by 10% acrylamide denaturing gels (SDS-PAGE) and transferred to membranes. The membranes were incubated with rabbit anti-protein kinase A (PKA) phosphorylation (1:1000, Millipore) and rabbit anti-cyclic AMP-response element binding protein (CREB) phosphorylation (1:1000, Santa Cruz, CA, USA). Then, the membranes were incubated with horseradish peroxidase-labeled goat anti-rabbit antibody (1:5000, Santa Cruz, CA, USA), and developed using an enhanced chemiluminescence detection kit (Millipore). After visualization, the blots were stripped by incubation in stripping buffer for 15 min, and then incubated with rabbit anti-PKA antibody (1:1000, Millipore) and rabbit anti-CREB antibody (1:1000, Santa Cruz, CA, USA). An internal control was performed using mouse anti-β-actin antibody (1:2000, Cell Signaling, Danvers, MA, USA). Western blot bands were scanned and analyzed with the ImageJ analysis software package (NIH).