Fig. 2.

Galectin-12 is a lipid droplet protein. (A) 3T3-L1 adipocytes were incubated for 2 h in serum-free DMEM. Protein levels of extracellular (in conditioned medium) and intracellular (cell-associated) galectin-12 and adiponectin were determined by Western blotting using specific antibodies. (B) Lipid droplets (LD) were purified from 3T3-L1 adipocytes by density gradient centrifugation, and the remaining cellular components were separated by differential centrifugation into five fractions containing plasma membrane (PM), high-density microsomes (HDM), low-density microsomes (LDM), cytosol, and mitochondria/nuclei (M/N). Levels of galectin-12, Glut-4, and perilipin A were determined in each fraction by Western blotting with respective specific antibodies. Each lane represents samples from an equal number of cells. (C) Immunostaining of 3T3 adipocytes showing galectin-12 (red) and perilipin A (green, Lower) on lipid droplets (green, Upper). (D) 3T3-L1 cells were incubated in the presence (MDI+) or absence (MDI−) of the adipogenic hormone mixture to induce adipocyte differentiation. At indicated time points, cells were extracted for analysis of galectin-12 expression by Western blotting. (E) 3T3-L1 cells at various periods during differentiation were stained for galectin-12 (red), lipid droplets (green), and the nuclei (blue). Data are representative of three experiments with similar results.