Fig. 2. AAM-B is targeted to droplets. (A) HeLa cells were transfected with cDNA encoding C-terminally Myc-tagged AAM-B and grown for 15 hours in media containing 100 µM oleic acid to induce lipid droplets. The cells were fixed and processed for indirect immunofluorescence localization of Myc (1,4) and protein disulfide isomerase (5) or stained for lipids with Bodipy 493/503 (2). Most cells had Myc staining (1) restricted to the periphery of neutral lipid-positive (2) droplets, as shown in the merge (3). Other cells displayed Myc staining (4) around lipid droplets and, in addition, a reticular pattern distributed throughout the cell that co-localized with -PDI IgG (5), as shown in the merge (6). (B) HeLa cells grown on coverslips in the absence of oleate were transfected with cDNAs encoding wild-type, Myc-tagged AAM-B. At various times post-transfection, the cells on the coverslip were fixed and processed for immunofluorescence detection of Myc. The cells remaining in the dish were collected, lysed and processed for immunoblotting to detect Myc (inset). To quantify the distribution of AAM-B, images of stained cells were systematically acquired until 50 cells had been photographed for each time point. The cells were then scored for the presence of either lipid droplet alone, or droplet plus reticular ER staining patterns. We did not detect any expressing cells until after a 2 hour incubation. Each point is the average ±s.e.m. of three separate experiments. Scale bars: 5 µm.