Lipid droplet isolation and subcellular fractionation

Lipid droplets were isolated as previously described with minor modifications [125]. In brief, cells were harvested in cold PBS, resuspended in hypotonic sucrose buffer (0.25 M sucrose, 1 mM EDTA supplemented with 1 mM DTT, 1x protease inhibitor cocktail and 1 mM PMSF) and lysed mechanically in a Dounce homogenizer. Post-nuclear supernatants (PNS) were overlaid with isotonic potassium phosphate buffer (0.1 M potassium phosphate pH 7.4, 100 mM KCl, 1 mM EDTA, supplemented with 1 mM PMSF) and centrifugation was performed for 2 h at 100 000 x g, 4°C in an SW60 rotor (Beckman Coulter). Floating lipid droplets were harvested using a bent canula, and PNS and lipid droplet fractions were analyzed by immunoblotting.

For subcellular fractionation, lipid droplets were separated from the microsomal fraction by differential centrifugation as previously described with minor modifications [126,127]. Briefly, cells were resuspended in hypotonic sucrose buffer, lysed mechanically in a Dounce homogenizer, and the resulting cell homogenate was centrifuged twice for 5 min at 600 x g, 4°C to separate the nuclei. To separate the mitochondria, the post-nuclear supernatant (PNS fraction) was centrifuged twice for 10 min at 10 000 x g, 4°C. The supernatant was transferred to an ultracentrifugation tube and overlaid with isotonic potassium phosphate buffer. Ultracentrifugation was performed in an SW60 rotor (Beckman Coulter) for 1 h at 100 000 x g, 4°C. The floating lipid droplet fraction was harvested as described above. The pelleted microsomal fraction was resuspended in RIPA lysis buffer (50 mM Tris, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 1x protease inhibitor cocktail, 100 mM PMSF, and 2% Triton-X-100 to ensure complete solubilization of the ER-retained HCV core^SPMT protein. For each fraction, equal amounts of total protein were subjected to immunoblot analysis.