Protease inhibitor and phosphatase inhibitor cocktail

Cells (5×10⁵ or 1×10⁶) were lysed in protein lysis buffer (2% SDS, 50 mM Tris-HCl, pH 7.5) containing protease inhibitor and phosphatase inhibitor cocktail (Roche, Basel, Switzerland) 26 (link). Protein concentration was determined by using a Bradford assay kit (BioRad, Hercules, CA, USA). An amount of 50-80 µg protein lysates were separated on 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes, which were blocked with 5% milk in Tris-buffered saline (TBS) for 1 h at room temperature, and then incubated with primary antibody overnight at 4 °C. After washing with TBST buffer (TBS with 0.05% Tween X100), the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratory, West Grove, PA, USA) for 2 h at room temperature and then revealed using enhanced chemiluminescent (ECL) reagent (Advansta, San Jose, CA, USA). Image and emission signal density measurements were quantified by using a BioSpectrum Image System (UVP, Upland, CA).

PCP was performed as previously described^{23 (link)}. For differentiated SGBS and hAPC cells, 5 × 150-mm dishes of cells at day 20 after differentiation, and for preadipocyte gradients, 3× T175 flasks of SGBS cells 1 day after confluency, were used per replicate. Cells were homogenized with a tissue homogenizer on ice in a 1:1 mixture of scraped cells to lysis buffer (20% sucrose, 20 mM Tris, pH 7.4, 0.5 mM EDTA, 5 mM KCl, 3 mM MgCl₂, protease inhibitor and phosphatase inhibitor cocktail (Roche)). Then, 2 ml supernatant was loaded onto a continuous 11 ml 20–55% sucrose gradient in 20 mM Tris, pH 7.4, 0.5 mM EDTA, 5 mM KCl and 3 mM MgCl₂. Organelles were separated at 100,000g (Beckmann, Rotor SW40 Ti) for 3 h at 4 °C. To isolate LDs, the 1-ml top fraction was cut with a tube-slicer (Beckman Coulter). The underlying 0.5-ml fractions were collected from the top to the bottom of the gradient.

Cells were harvested and proteins were extracted using lysis buffer (50 mM Tris pH 7.5, 0.15 M NaCl, 50 mM NaF, 5 mM EDTA, 1 mM EGTA 0.1 mM Na3VO4 0.1% Triton X-100) containing Protease Inhibitor and Phosphatase inhibitor Cocktail (Roche, Diagnostics; Mannheim, Germany), incubated on ice for 20 min and cleared by microcentrifugation. Protein concentrations of cellular lysates were measured by the BioRad™ protein assay kit (Richmond, CA, USA). Equal amounts of protein (20 μg) were loaded in each lane with loading buffer containing 25% 0.25 M Tris-HCl, pH 6.8, 50% glycerol, 10% SDS, 5% mercaptoethanol and 0.025% bromophenol blue. Samples were boiled for 5 min before being separated on 8–10% SDS-PAGE gels, depending on the protein to be analyzed. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (BioRad) using an electrophoretic transfer system (Bio-Rad, Hercules, CA, USA). The membranes were then incubated overnight at 4 °C with specific primary antibodies. After washing to remove unbound antibodies, horseradish peroxidase-linked goat anti-mouse or goat anti-rabbit IgG secondary antibodies were added at a dilution ratio of 1:5000 and the membranes were incubated at room temperature for 2 h. The immune complex was visualized with an ECL system (Cell Signaling Technology, Danvers, MA, USA).