Membrane protein extraction

Membrane protein extraction assay was conducted by the Mem-PER Plus Membrane Protein Extraction Kit (Thermo Scientific, catalog: 89842) and according to manufacturer’s protocol with minor modification. Cortical neuron cultures at DIV 14 or whole brains were harvested in 1 mL of Cell Wash Solution. After washing and a centrifuge at 300×g for 5 min, cell or tissue pellet was resuspended in 500 μL of Permeabilization Buffer. The cell or tissue suspension was then incubated at 4 °C for 10 min with constant rotation. Permeabilized cells were subjected to a centrifuge for 15 min at 16,000×g at 4°C. The supernatant containing cytoplasmic proteins was transferred to a clean tube and saved as cytoplasmic fractions. The pellet was resuspended in 300 μL of Solubilization Buffer by pipetting up and down, followed by an incubation of 10 min at 4°C with constant rotation. After another centrifuge at 16,000×g for 15 min at 4℃, the supernatant containing solubilized membrane and membrane-associated proteins was collected.