Sw38 rotor

NMuMG cells, grown to 80–90% confluence in two 150 mm plates, were washed with ice-cold PBS, and harvested by scraping in 20 mM HEPES pH 7.4, 1 mM EDTA, 250 mM sucrose, 10 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF, and complete protease inhibitor cocktail (Roche). The separation of the high density microsomal (HDM) and low density microsomal (LDM) fractions was adapted from published work (84 (link)) with some changes. The cells were homogenized with 25 to 30 strokes using a tight-fitting Dounce homogenizer, and the homogenates were centrifuged at 500 rcf for 10 min at 4°C to remove unbroken cells. Equal supernatant protein amounts were centrifuged at 10,080 rcf for 20 min at 4°C. The cytosol (supernatant) fraction was then centrifuged using Sorvall RC6T at 15,750 rcf and 4°C for 20 min, and the resultant supernatant was re-centrifuged in 70Ti rotor (Beckman) at 175,000 rcf for 120 min at 4°C to separate the cytosol and pelleted LDM fractions. The resulting pellets (LDM) were resuspended in extraction buffer, loaded on a layered 10 – 36 % (wt/wt) discontinuous sucrose gradient cushion, and centrifuged using SW38 rotor (Beckman) for 12 h at 4°C at 284000 rpm. Twelve fractions were collected, starting from the top of the gradient, and equal volumes of each fraction were analyzed by SDS-PAGE followed by immunoblotting.