Figure 2
From: Morphologically and Functionally Distinct Lipid Droplet Subpopulations
Subpopulation of lipid droplets with different sizes and protein compositions.
Total LDs and cytosol were isolated from CHO K2 cells. The total LDs were further fractionated by size using differential centrifugation (Materials and Methods). LD fractions 1, 2 and 3 were collected. (A) LD size of the subpopulations. LD fractions were processed for fixation, dehydration and infiltration and finally embedded in Embed 812. Then the ultra-thin sections of the three fractions were prepared and analyzed by EM. Bar = 1 μm. (B) ER and mitochondrial markers in the subpopulations. Proteins of these fractions were extracted with chloroform:acetone (300 μl:700 μl), separated using SDS-PAGE, analyzed by Western blotting (equal amount of protein per lane) with the indicated antibodies and stained by Colloidal Blue. (C) Protein recruitment to the subpopulation. These three fractions were incubated with Buffer B (Con) or cytosol plus 1 mM GTPγs (C + G) for 1 h at 37 °C. LDs were then re-isolated and the proteins were extracted with acetone and analyzed by Western blotting with the indicated antibodies. (D) Protein composition of the subpopulations. The proteins of the fraction 1 and 3 were extracted with chloroform:acetone (300 μl:700 μl) and analyzed by silver staining and Western blotting (equal amount of protein per lane) with the indicated antibodies.
