Biii tubulin

The hippocampal slices were obtained from 8-to 12-week-old C57BL/6 male mice killed by decapitation. The temporal lobes were cut into 350-mm-thick slices with vibratome. The hippocampal slices were then cultured for immunoblotting in MEM media (Corning) and treated with 5-mM GlcNAc and 150-mM UDP-GlcNAc or 50-mg/mL Ab 25e35 for 1 week. Slices were then homogenized in RIPA buffer containing protease inhibitors (Roche Diagnostics). After 2 hours at 4 C, homogenates were centrifuged at 4000 rpm to discard cellular debris. Protein content was determined by Bradford Assay (Sigma-Aldrich). Protein lysates were diluted in Laemli buffer, boiled at 90 C for 5 minutes, and then resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred onto the nitrocellulose membrane (Bio-Rad), which were blocked with Tris-buffered saline and 0.1% Tween 20% and 10% nonfat dry milk (GE-Healthcare). Membranes were then incubated at 4 C overnight with the following primary antibodies: syntaxin (Abcam), synaptophysin (Millipore), bIII tubulin, and actin (Sigma-Aldrich). Appropriate secondary IgG HRP-conjugated (GE Healthcare) was added for 1 hour, and chemiluminescent detection was performed with ECL Plus advanced (Amersham and GE Healthcare). Quantitative analysis of the signal obtained was performed by Image J software (National Institutes of Health).