Biochemical assays and evaluation

Whole mouse brains were homogenized with a Dounce tissue grinder (20

times) in neuronal protein extraction reagent (N-PER) (Thermo Scientific)

containing protease/phosphatase inhibitors (Cell Signaling #5872).

Triton X-100 (Sigma #X100-100ML) was added at

a final concentration of 1%, and the samples were incubated with rotation for 1

hour at 4 °C.

Samples were centrifuged at 10,000 × g g

for 10 min at 4 °C, and the supernatant was collected.

For Neuro2A lysates, cells were washed with 1X PBS 3 times and incubated

for 5 min on ice in the presence of N-PER reagent supplemented with protease

inhibitors.

Samples were centrifuged at 10,000 × g g

for 10 min at 4 °C to remove cellular debris.

Total protein

concentration was measured in brain and Neuro2A lysates (D^TM‱ Protein Assay Kit II, Biorad), and samples were

used in subsequent experiments.