Cell culture media from HEK-293 cells were collected and cleared from insoluble material by centrifugation for 10 min at 16,000×g, 4 °C. Cells were washed with PBS and extracts were prepared by scraping and vortexing the cells in lysis buffer composed of 20 mM Tris–HCl, pH 7.5, containing 150 mM NaCl, protease inhibitor cocktail (complete, Mini, EDTA-free, Roche), PhosSTOP phosphatase inhibitor cocktail (Roche) and Triton X-100 to a final concentration of 1%. Extracts were cleared from insoluble material by centrifugation for 10 min at 16,000×g, 4 °C.
For protein extraction from mouse tissues, hippocampi from 64 week-old mice (4–5 animals per group) were homogenized in 200 µl of 20 mM Tris–HCl, pH 7.5, containing 150 mM NaCl, protease inhibitor cocktail (complete, Mini, EDTA-free, Roche) and PhosSTOP phosphatase inhibitor cocktail (Roche) using a dounce homogenizer as previously described3 (link). Proteins were solubilized by addition of Triton X-100 to a final concentration of 1%. Extracts were cleared from insoluble material by centrifugation for 30 min at 20,000×g, 4 °C. Protein concentration was determined with Quick Start Bradford 1 × Dye Reagent (BioRad Laboratories) as described by the manufacturer. n = 4–5 and three technical replicates were performed.
For protein extraction from mouse tissues, hippocampi from 64 week-old mice (4–5 animals per group) were homogenized in 200 µl of 20 mM Tris–HCl, pH 7.5, containing 150 mM NaCl, protease inhibitor cocktail (complete, Mini, EDTA-free, Roche) and PhosSTOP phosphatase inhibitor cocktail (Roche) using a dounce homogenizer as previously described3 (link). Proteins were solubilized by addition of Triton X-100 to a final concentration of 1%. Extracts were cleared from insoluble material by centrifugation for 30 min at 20,000×g, 4 °C. Protein concentration was determined with Quick Start Bradford 1 × Dye Reagent (BioRad Laboratories) as described by the manufacturer. n = 4–5 and three technical replicates were performed.
Free full text: Click here
