Isolation and Characterization of Mouse Brain Membrane Proteome

All mouse studies were performed following protocols that received approval from the Indian Institute of Science Education and Research Pune Institutional Animal Ethics Committee. The mice were genotyped using an established protocol (5 (link)). The mouse brain membrane proteomes were prepared using a previously described procedure (15 (link)). Briefly, the mice were first anesthetized with isoflurane and euthanized by cervical dislocation, following which the brains of the mice were harvested. A fresh half brain was suspended in 500 μl of cold sterile DPBS and homogenized using a tissue homogenizer (Bullet Blender 24, Next Advance) with one scoop of glass beads (0.5-mm diameter; Next Advance) at a speed setting of 8 for 3 min at 4 °C. To the brain homogenate, an additional 500 μl of cold sterile DPBS was added, mixed by pipetting, and centrifuged at 1000 × g for 5 min at 4 °C to separate the tissue debris. The resulting supernatant (∼700 μl) was separated and centrifuged at 100,000 × g for 45 min at 4 °C. The resulting supernatant was discarded, and the pellet (membrane proteome) was washed with cold sterile DPBS (three times) and resuspended in 1 ml of cold sterile DPBS by pipetting. The protein concentration of the brain membrane proteome was estimated using BCA protein assay kit (Pierce).