Identification of AMN as a protein colocalizing with and binding to cubilin. (A) Top panels: Immuno-peroxidase staining of AMN (left) and cubilin (right) in human kidney proximal tubules. Bottom panels: Immunoelectron staining with 10-nm gold particles of AMN (left) and cubilin (right) in the dense apical vesicles and plasma membrane of proximal tubules known to function in membrane receptor recycling.32 Original magnification, × 1000 (top panels) and × 46 000 (bottom panels). (B) Detergent-solubilized human kidney cortex membrane proteins were purified by IF-cobalamin affinity chromatography, separated on a 4% to 16% SDS-PAGE gel (lane 1, Coomassie stain), and subjected to anti-AMN immunoblot (lane 2). Also shown are anti-AMN immunoblots of IF-cobalamin–binding proteins from the supernatant fraction of kidney membranes prior to detergent solubilization (lane 3), human urine (lane 4), and kidney cortex membranes before (lane 5) and after (lane 6) PNGase-F digestion. (C) Anticubilin and anti-AMN immunoblots of Superose-6 gel filtration fractions of human kidney cortex IF-cobalamin–binding proteins eluted in PBS, pH 7.4, with 2 M or 6 M urea. Cubilin and AMN coeluted in fractions 9 and 10 in 2 M urea but separated in 6 M urea, where AMN elutes in fractions 16 and 17.
