Preparation of Transgenic Mouse Brain Homogenates

Hemi-sected transgenic mouse brains were stored at −80 °C prior to homogenization. Frozen brains were placed onto a chilled metal block on dry ice and were transected coronally at the level of the midbrain to separate caudal and rostral brain sections (see also Fig. 15). Sections were placed in 1 mL PBS prepared with protease inhibitor tablet (Roche) and were sonicated using a probe sonicator (Omni Sonic Ruptor 400) at 30% pulse/30% power, 20 times, 10-s cycles. The resulting homogenates were centrifuged at 10,000 xg for 15 min at 4 °C. and supernatants used for subsequent steps [27 (link)]. Clone 1, Clone 9, and Clone 10 lysates were prepared as described previously [27 (link)]. Cell pellets were lysed in 0.1% Triton-X/PBS solution plus protease inhibitor cocktail (“Complete”, Roche) and were clarified with sequential five-minute 500 xg and 1000 xg spins. Protein concentrations in brain homogenates and cell lysates were measured by Bradford assay (Bio-Rad) and were normalized to 5 μg/μL and stored at −80 °C until use.