The glycosylation patterns of cellular and secreted HJV in HEK293 and Hep3B cells. (A) HEK293 cells were transiently transfected with wild-type HFE2-pcDNA3 using Lipofectamine 2000 (Invitrogen) in 6-well plates and incubated in complete medium. Approximately 48 hours after transfection, serum-free medium (1 mL) was added. After an additional 24 hours of incubation, conditioned medium was collected and cell lysate was prepared using 300 μL NET-Triton buffer with Protease inhibitor cocktail. Proteins in the medium (1 mL) were precipitated using 6% TCA. Proteins from both the cell lysate (Lysate) and medium were equally divided into 3 parts and were subjected to Endo H and PNGase F digestion followed by detection of HJV by immunoblot. (B) Hep3B cells were transiently transfected with HFE2-pcDNA3 using LipoFectamine 2000. The transfection of HFE2 into Hep3B cells, Endo H and PNGase F digestion, and immunodetection were performed as described for HEK293 cells. There was a better separation on the gel for Hep3B cells, which accounts for the apparent increased separation between the control and digested samples in this cell type. The data are representative of 3 experiments for each cell type.