Cubilin expression in CHO cells transfected with wild-type or c.1113-1145del AMN cDNA. (A) CHO cell lines expressing a “mini-cubilin” construct of rat origin were additionally transfected with wild-type (WT) or c.1113-1145del (mut) canine AMN cDNA constructs, and stable double transfectants expressing cubilin and AMN were selected. Nonpermeabilized cells were stained for confocal fluorescence microscopy by incubation at 4°C with anticubilin and subsequently with labeled anti-rabbit IgG. Abundant surface staining of cubilin was observed in cells expressing wild-type, but not mutant, AMN. (B) Proteins isolated by IF-cobalamin pull-down from lysates of double-transfectant cell lines were subjected to endo H or mock digestion followed by SDS-PAGE and Western blot with anticubilin serum. Golgi maturation of cubilin N-linked oligosaccharides to endo H-resistant forms was observed in cells expressing wild-type AMN, but not in cells expressing the mutant AMN. (C) Lysates of double-transfectant cell lines were subjected to Western blotting with anti-myc, confirming expression of wild-type and mutant AMN.