Fig. 2.

Expression of KIAA1363 lowers RE levels in COS-7 cells. A: Schematic representation of pulse-chase cell experiment. B, C: COS-7 cells were seeded in 12-well plates and transfected with plasmids encoding HIS-tagged mKIAA1363 or LacZ (control). B: Protein expression levels of HIS-tagged recombinant proteins were analyzed by Western blotting (arrows indicate bands corresponding to KIAA1363 protein). C: COS-7 cells expressing LacZ or mKIAA1363 were cultured with DMEM supplemented with 10% FCS and antibiotics (=basal). Then, cells were incubated in DMEM containing 10% FCS, oleic acid (300 μM), and ROH (40 μM) for 24 h (=loaded/pulse). After the loading period, cells were incubated with DMEM low glucose (1 g/l) containing 2% FA-free BSA for 8 h (=serum starvation/chase). Lipids were extracted with n-hexane:2-propanol (3:2, v/v). RP content was determined by HPLC-FD and normalized to milligram cell protein. Data are mean + SD and representative for two independent experiments (n = 3). Statistically significant differences were determined by Student's unpaired t-test (two-tailed: ∗P < 0.05).