Cubilin and AMN expression in normal and I-GS affected dog kidney in vivo. (A) Membrane fractions prepared in bulk from normal (N) and GS kindred affected (A) dog kidney cortex were detergent solubilized and subjected to IF-cobalamin affinity chromatography. Eluted proteins were concentrated, separated by SDS-PAGE on an 8% to 16% gradient gel, and silver stained. Two bands missing from the affected dog preparation migrated at approximately 48 and 40 kDa (arrowheads) and comigated with anti-AMN immunoreactive bands on Western blots of duplicate gels. They were identified by LC/MS/MS peptide mapping as full-length and C-terminally truncated AMN, respectively. (B) Anti-AMN Western blots of proteins isolated by large-scale IF-cobalamin affinity chromatography from normal dog kidney (lane 1) or by small-scale IF-cobalamin pull-down from kidney cortex homogenates of a normal dog (lane 2), a GS kindred affected dog (lane 3), and an AS kindred affected dog (lane 4). (C) Immunoperoxidase staining of cubilin in renal cortex of an unrelated normal dog (left) and I-GS affected dogs of the GS (middle) and AS (right) kindreds expressing AMN mutations c.1113-1145del and c.3G>A, respectively. Total original magnification, 200 ×.