Extraction and preparation of enzyme sources for in vitro enzymatic assay

Human DGAT1, DGAT2 and glycerol-3-phosphate acyltransferase 1 (GPAT1) enzyme sources for in vitro enzymatic assay were prepared as described previously [27]. Human DGAT1, DGAT2 and GPAT1 proteins were produced in Sf9 insect cells by using the Bac-to-Bac Baculovirus Expression System. Sf9 insect cells were infected with recombinant baculovirus encoding each enzyme for about 3 days, harvested, and lysed by using Dounce homogenization in buffer A [10 mM Tris-HCl, 250 mM sucrose, 1 mM ethylenediamine-tetraacetic acid (EDTA), pH 7.4]. Total membrane fraction containing enriched human DGAT1 or DGAT2 was isolated by sequential centrifugations at 600 and 100,000 g and resuspended in buffer B (20 mM Tris-HCl, 250 mM sucrose, pH 7.4). For in vitro GPAT1 assay, the homogenate was sequentially centrifuged at 600 and 8,000 g and the collected crude mitochondrial membrane fraction was resuspended in buffer B. The protein concentration was measured using the Bradford protein quantification assay.