Figure 7.

Secreted Pmel17 is fully Golgi modified, and the N-glycan in GAP3 contributes to the secretion. A) MNT-1 cells and HeLa cells overexpressing Pmel17-i were radiolabeled and chased for 1 h (cell lysates of MNT-1 cells) or 4 h (HeLa cells and medium of MNT-1 cells). Cell lysates and medium were immunoprecipitated with NKI/beteb. Immunopurified samples were split into 3 portions; one was digested with EndoH (750 U), another was digested with PNGaseF (750 U), and the third was the untreated control. Digestion reactions were performed for 12 h at 37°C. Digested samples were electrophoresed and visualized by autoradiography. B) MNT-1 cells were radiolabeled and chased in the presence of DMSO, Tun at 2 μg/ml, or DMM at 1 mM for 4 h. Cell lysates and medium were immunoprecipitated with NKI/beteb, separated by electrophoresis, and visualized by autoradiography. Intensities of specific bands, Mα, relative to control are quantified (bottom panel). Graphed data are mean ± sd ratios of Mα intensity in medium/cells from 3 independent experiments. C) HeLa cells overexpressing S83A, T108A, S113A, S83A/T108A/S113A, or S570A were radiolabeled and then analyzed as described above.