Effect of AMN on cubilin expression and processing in CHO cells. (A) Cubilin (amino acids 1-1389) and AMN (full length plus myc epitope) constructs encoded by plasmid cDNA used for stable transfection of CHO cells. The position of the IF-cobalamin (IF-cbl) binding site (CUB domains 5-8)6 is indicated and the truncated CUB domains 9-27 are shown by dashed lines. (B) Immunoblots of cubilin and AMN in cell lysates (lanes 1-3) and endo H digestion of IF-cobalamin affinity-purified cubilin from cells transfected with cubilin, AMN, and cubilin/AMN cDNA. Gel mobility shifts indicate that cubilin oligosaccharides in single transfectants are endo H sensitive (compare lanes 4 and 6), but the bulk of cubilin oligosaccharides in cubilin/AMN double transfectants are endo H resistant (compare lanes 5 and 7).
