Cell-associated HJV has high-mannose oligosaccharides, whereas cellular neogenin has complex oligosaccharides. (A) Endo H and PNGase F sensitivity of cellular HJV and cellular neogenin. Cell lysate was collected from HJV-HepG2 (HJV) cells (transfected with a nonspecific siRNA) or HJV-G320V-HepG2 (HJV-G320V) cells. Lysates were subjected to Endo H and PNGase F digestion. Immunoblots were performed using anti-neogenin (0.4 μg/mL; Santa Cruz Biotechnology) and anti-HJV antibodies (0.22 μg/mL). (B) Analysis of Endo H sensitivity of neogenin and HJV by pulse chase (in neogenin/HJV-HEK293 and HJV-HepG2 cells, respectively). Metabolic labeling and immunoprecipitations were performed as described previously³⁹  with the following modifications: HJV-HepG2 (bottom panel) or neogenin/HJV-HEK293 (top panel) cells in 35-mm dishes were labeled in 1 mL Met/Cys-free media with 100 μCi ³⁵S-(Met/Cys) for 30 minutes. Cells were then washed and incubated in unlabeled medium for the time points indicated. Immunoprecipitations were performed using 2 μL rabbit anti-HJV antibody, 18745 (generated against residues 1-401 of HJV as described previously¹⁸ ), or rabbit anti-neogenin 21567 antibody, which was generated using the neogenin ectodomain as an antigen (purified as described previously¹⁶ ) to generate a polyclonal antibody in rabbits (Pocono Rabbit Farm & Laboratory, Canadensis, PA). Immunoprecipitated proteins were subjected to control (mock) or Endo H digestion and separated by SDS-PAGE, followed by soaking of the gel in Amplify (GE Healthcare, Chalfont St Giles, United Kingdom) and drying of the gels before exposure to film. (C) Analysis of time taken for HJV to traffic to the cell surface after induction of HJV expression. tTA-HJV-HepG2 cells in 60-mm dishes were induced to express HJV by addition of 2 μg/mL doxycycline for 0, 2, 3, 4, 5, and 6 hours. Cell-surface proteins were biotinylated at 4°C and pulled down using streptavidin agarose. A total of 100% of the total biotinylated proteins and 15% of the internal (nonbiotinylated) proteins were subjected to immunoblotting for HJV using a rabbit anti-HJV 18746 antibody. This experiment was repeated once with similar results. (D) Cell-surface HJV has high-mannose oligosaccharides. Biotinylation of cell-surface proteins was conducted as described previously.⁴⁰  Briefly, HJV-HepG2 cells in a 6-well plate at approximately 80% confluence were biotinylated with 0.25 mg/mL Sulfo-NHS-Biotin (Thermo Electron, Waltham, MA) at 4°C for 30 minutes. Cells were immediately solubilized in NET-Triton/1× Protease inhibitor cocktail; then biotinylated proteins were isolated using streptavidin agarose beads (Thermo Electron). Bound proteins were eluted with NET-Triton/1% β-mercaptoethanol/0.5% SDS and subjected to digestion with Endo H and PNGase F (New England Biolabs), followed by immunodetection of HJV and neogenin. Data are representative of 3 independent experiments.