Isolation and Characterization of Cellular Organelles

Cell lysates were prepared from primary cultured microglia, neurons, HEK293T cells or brain tissue by RIPA lysis buffer (Beyotime, China) with protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche). Nuclear and cytoplasmic extracts were prepared by the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, #78833) according to the manufacturer's instructions. Protein concentrations were determined using an enhanced bicinchoninic acid (BCA) protein assay kit (Beyotime, China) prior to Western blot or immunoprecipitation analysis. Lysosomes were isolated by a lysosome isolation kit (Sigma) with density gradient ultracentrifugation according to the manufacturer's description. Brain tissue was prepared from the ipsilateral parietal cortex (penumbra of the middle cerebral artery) or hippocampus and the corresponding area of the contralateral side 20 (link).