Western blot analysis

Protein samples of hippocampal tissue were prepared and homogenized in cold lysis buffer containing 25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% nonyl phenoxypolyethoxylethanol, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, (Thermo Scientific, Waltham, MA), and protease and phosphatase inhibitor cocktail solutions (GenDEPOT, Barker, TX). Homogenates were centrifuged at 20,000 g for 30 min at 4°C, and the supernatants were harvested and stored at −70°C. Protein concentrations were determined using the BCA assay (Thermo Scientific). Protein samples (40 μg) were then separated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane, which was subsequently incubated in primary antibodies against interleukin-1β (IL-1β; Millipore Corporation, Billerica, MA), cox-2 (Abcam, San Francisco, CA), RAGE (Abcam), Ang-II (Abcam), interleukin-6 (IL-6; Abcam), ERK (Cell Signaling, Danvers, MA), p38 MAPK (Cell Signaling), c-Jun N-terminal kinases (JNK; Cell Signaling), or ChAT (Millipore Corporation). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnologies, Santa Cruz, CA) was used as an internal control. A goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Cell Signaling) was then added. Detection was carried out using an enhanced chemiluminescence system (Thermo Scientific) with a Lumino Image Analyzer (Las-4000; Fujifilm, Tokyo, Japan). Densitometry was performed for specific markers normalized to GAPDH using Multigage software (Fujifilm).