FIGURE 2.
Expression of FLAG-tagged DGAT1 constructs in HEK-293T cells. A, an immunoblot shows transient expression of the FL-DGAT1 constructs described in Fig. 1C in HEK-293T cells. Total cellular membranes (50 μg of protein) were immunoblotted with anti-FLAG and anti-calnexin antibodies. B, shown is in vitro DGAT activity in membranes from cells expressing FL-DGAT1 constructs. Membranes from cells were assayed for in vitro DGAT activity by measuring the formation of [14 C]TG from 1,2-diacylglycerol and [14C]oleoyl-CoA. The activities of the various mutants are normalized to that of FL-DGAT1. The activities for untransfected cells and cells transfected with FL-DGAT1 were 7.4 and 128 pmol/min/mg of protein, respectively. Data are from one experiment, performed in duplicate, which was repeated twice with similar results.