Secreted HJV has complex oligosaccharides and is derived from the pool of HJV at the plasma membrane. (A) Secreted HJV has complex oligosaccharides. Conditioned medium was collected from HJV-HepG2 cells and subjected to Endo H and PNGase F digestion, followed by a Western blot using a rabbit anti-HJV antibody. Conditioned medium from control-HepG2 cells (C) was included as a negative control. (B) Chasing the release of biotinylated cell surface HJV. Cell-surface HJV in HJV-HepG2 cells was biotinylated at 4°C, followed by incubation at 37°C for 4 or 8 hours in complete medium. The total biotinylated HJV in the cell lysate (HJV-HepG2 biotinylated) and the biotinylated HJV released into the medium were isolated using streptavidin-agarose beads. The eluates were digested with Endo H and PNGase F. Endo H and PNGase F digestion of one-third of cell lysates are also included. Lysate (Ctrl L) or medium (Ctrl M) from Control-HepG2 cells were used as negative controls. Experiments were repeated 3 times.