Neogenin is necessary for sHJV release but not for the retention of high-mannose oligosaccharides by HJV. (A) Knockdown of neogenin does not alter the glycosylation of cellular HJV. Endogenous neogenin in HJV-HepG2 cells was knocked down using a siRNA specific for neogenin (for the control HJV-HepG2 cells transfected with a nonspecific control siRNA; see Figure 2A). siRNA specific for human neogenin (25 nM; Dharmacon RNA Technologies, Lafayette, CO) or scrambled control siRNA was transfected twice (once on day 1 and once on day 3) using the RNAiMAX transfection reagent (Invitrogen). Seventy-two hours after the second transfection, cell lysates were subjected to Endo H and PNGase F digestion. Both neogenin and HJV were detected by immunoblot using antineogenin and anti-HJV antibodies, respectively. (B) Disruption of the HJV/neogenin interaction inhibits release of HJV but does not perturb the high-mannose glycosylation of HJV. HJV-HepG2 cells were treated with soluble neogenin ectodomain fragments consisting of the FNIII repeats 1-6 (1 μM) or a smaller fragment of only repeats 5 and 6 (40 nM) in serum-free medium overnight. Soluble neogenin FNIII 1-6 and FNIII 5-6 as well as the whole neogenin extracellular domain (ectodomain) were generated as previously described¹⁶  and were a gift from F. Yang and P. J. Bjorkman at CalTech. Conditioned medium and cell lysates were collected, followed by Endo H and PNGase F digestion and immunodetection of HJV. Untreated HJV-HepG2 cells were used as a control (C). 1-6 indicates neogenin FNIII 1-6 fragment; 5-6, neogenin FNIII 5-6 fragment. *A nonspecific band resulting from cross reaction of either the primary or secondary antibodies with the FNIII 1-6 fragment. (C) Neogenin ectodomain inhibits HJV release and results in HJV accumulation within cells. HJV-HepG2 cells in 12-well plate were incubated in complete medium with or without addition of soluble neogenin ectodomain at 1 μM. After 24 hours of incubation, the total cell lysate (L) and 20% of conditioned medium (CM) were subjected to detection of HJV by immunoblot. Actin in cell lysates was used as a loading control. These data are representative of at least 2 experiments.