Specificity of mAbs CG3, LC1, and Pep3-43 and the detection of TM5b in human erythrocyte ghost membranes.

(A) Using SDS-PAGE, 2 μg each of rTM5, rTM5a, and rTM5b and 30 μg human erythrocyte ghost membranes were separated on a 10% gel. Three identical sets of proteins were transblotted onto nitrocellulose membranes and probed either with mAbs CG3^1:1000 (panel labeled “mAb CG3”), LC1^1:500 (panel labeled “mAb LC1”), or Pep3-43^1:100 (panel labeled mAb “Pep3-43”). Secondary antibodies conjugated with HRP were used, and the signals were developed enzymatically. Molecular weight standards (lane 1) are phosphorylase b (97 kd), bovine serum albumin (66 kd), ovalbumin (45 kd), carbonic anhydrase (31 kd), and soybean trypsin inhibitor (21 kd). (B) This panel demonstrates the presence of TM5b, but not TM5a, in human erythrocyte ghost membranes. Using SDS-PAGE, we separated 2 μg each of purified rTM5a (lanes 1 and 5) and rTM5b (lanes 2 and 6) and 30 μg ghost membranes prepared from rat (lanes 3 and 7) and human (lanes 4 and 8) erythrocytes on an 10% gel in the absence (panel labeled “No Urea”) and presence (panel labeled “20% Urea”) of 20% urea. Both transblots were probed with mAb Pep3-43^1:100 and an HRP-conjugated secondary antibody against mouse IgG; the color was developed enzymatically. The arrow points to the band that comigrated with rTM5b in human and rat ghost membranes. (C) Amino acid (1 letter code) sequences of hTM5,35 rTM5,25 rTM5a, and rTM5b36are shown; numbers on the top refer to the amino acid residues. The − indicates residue identical with those in hTM5, with human (h) and rat (r) identified. rTM5a is identical to rTM5b except for the boxed region (residues 152-177) encoded by an alternative exon.