Fig. 4. Identification of ERK2 as an activator of lipid droplet formation in the cell-free system. (A) Hydrophobic interaction chromatography (which was the final step in the partial purification of the cytosolic activator for the formation of lipid droplets) on a Resource Phe^TM column using the Äkta Prime system (see Materials and Methods), starting from rat adipocyte cytosol (200 animals) (Marchesan et al., 2003). (B) The ability of the fractions 1-3 recovered from the Resource Phe^TM column, to activate the assembly of lipid droplets in the cell-free system (as percentage of the total amount of newly formed triglycerides that are assembled into lipid droplets). (C) Fraction 3, containing the major amount of activity and the least amount of protein (based on the absorption at 280 nm) was collected, concentrated and electrophoresed in a 10% SDS-polyacrylamide gel and stained with Sypro Ruby protein stain. The protein bands (figures on the right-hand side of the gel) were cut out, digested with trypsin and analyzed by MALDI-TOF or, when no hit was obtained, by MS/MS. For the identification of the proteins, see text. The figures on the left-hand side are the molecular mass markers. (D) The ability of recombinant proteins corresponding to ERK2 (0.4 µg/assay) and adiponectin (0.5 µg/assay), or purified citrate lyase (1 U/assay), to activate the assembly of lipid droplets in the cell-free system. Results are given as the percentage of newly formed triglycerides that are assembled into lipid droplets subtracted from the buffer blank (mean ± s.d.; n=3).