Immunodetection and characterization of AMN in canine kidney. AMN was enriched from membrane (lanes 1-4) or supernatant (lane 5) fractions of homogenates of normal dog kidney cortex by IF-cobalamin affinity column chromatography. Eluted proteins were separated by SDS-PAGE (lanes 1-3) or by 2-dimensional gel electrophoresis (blots 4 and 5). Proteins in lanes 1 and 2 were subjected to mock and PNGase F digestion, respectively, prior to electrophoresis and blotting. The additional, lightly stained band in lane 2 migrates at the expected position of PNGase F. Antiserum against the extracellular domain peptide A, as shown in the schematic, was used for immunodetection in 1, 2, 4, and 5, and antiserum against the cytoplasmic domain peptide B was used in lane 3. The schematic of the mature, full-length AMN protein indicates the relative positions of the site of N-linked oligosaccharide addition (NXT/S), the cysteine-rich domain (CRD), and peptides A (amino acid residues 340-353) and B (residues 388-400) against which antisera were raised. The excess positive charge of the intracellular domain protein sequence including peptide B is + 3. Two vertical lines indicate the single transmembrane domain.