Total akt

Brain tissues were homogenized in 20mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid containing 0.32M sucrose and centrifuged at 700 g for 10 minutes at 4°C. The supernatants were centrifuged again at 14,000 g for 10 minutes at 4°C, and the pellets were resuspended in T-PER reagent (Thermo Scientific, Rockford, IL). Proteins were separated and transferred onto a nitrocellulose membrane. Blots were immunostained overnight at 4°C with primary antibody against total GSK-3β (BD, Franklin Lakes, NJ), phospho-GSK-3β at Ser9, total Akt (the serine/threonine kinase, also known as protein kinase B or PKB), phospho-Akt at Ser473, total extracellular signal-regulated kinases (ERKs), phospho-ERK at Thr202/Tyr204, total mTOR, phospho-mTOR at Ser2448, total P70S6 kinase (P70S6K), phospho-P70S6K at Thr389, total eukaryotic elongation factor-2 (eEF2), phospho-eEF2 at Thr56, PSD95 (all from Cell Signaling, Beverly, MA), total tropomyosin-related kinase B (TrkB; Millipore, Billerica, MA), phospho-TrkB at Tyr817, or the house-keeping gene β-actin (Abcam, Cambridge, MA). Membranes were then incubated with secondary antibodies (LI-COR, Lincoln, NE) for 1 hour at room temperature. Finally, blotted proteins were detected and quantified using the Odyssey infrared imaging system (LI-COR).