Subcellular Fractionation of Mouse Midbrain and Cultured Cells

According to the manufacturer’s instructions, subcellular fractions were prepared using an endoplasmic reticulum isolation kit (Sigma-Aldrich). All procedures were performed at 4 °C. Mouse midbrains were isolated, cut into small pieces, and homogenized in four volumes of ice-cold Isotonic Extraction Buffer (10 mM HEPES, pH 7.8, 250 mM sucrose, 25 mM KCl, 1 mM EGTA, and 1× Protease and Phosphatase Inhibitor Cocktail) with Dounce homogenizer (12 strokes). Cultured cells were harvested, washed with ten volumes of PBS, and spun down at 600 × g for 5 min. The cell pellet was suspended and incubated in three volumes of ice-cold Hypotonic Extraction Buffer (10 mM HEPES, pH 7.8, 25 mM KCl, 1 mM EGTA, and 1× Protease and Phosphatase Inhibitor Cocktails) for 20 min to allow the cells to swell. Swollen cells were centrifuged at 600 × g for 5 min. The new cell pellet was homogenized in two volumes of ice-cold Isotonic Extraction Buffer with Dounce homogenizer (10 strokes). The homogenate (referred to as total lysate) from brain tissues or cultured cells was spun at 1000 × g for 10 min. The supernatant (S1) was collected, and the pellet (P1) was saved as crude nuclei fraction. S1 was centrifuged at 12,000 × g for 15 min. The supernatant (S2) was collected, and the pellet (P2) was saved as crude mitochondria fraction. S2 was further centrifuged at 100,000 × g for 60 min. The supernatant (S3) was collected as cytosol fraction, and the pellet (P3) was saved as ER microsomes fraction. Crude nuclei fraction, crude mitochondria fraction, and ER microsomes fraction were lysed in 1% SDS buffer. SDS was added to the total lysate and cytosol fraction to 1% final concentration. Equal amounts of protein from total lysate and each fraction were resolved in SDS-PAGE and applied to western blot analysis.