Distinct Epitopes on CD13 Mediate Opposite Consequences for Cell Adhesion

Figure 2

Inhibitory mAbs C and E recognize CD13 in monocytes and endothelial cells. (a) Lysates of U-937 cells were immunoprecipitated with mAbs 452, C, E or a control IgG, bound to protein A/G agarose beads. Immunoprecipitates were eluted from beads and resolved in 10% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and probed with mAb C (2 µg/ml) and HRP-conjugated secondary antibody. (b) U-937 cells, human peripheral blood monocytes, or endothelial HMEC-1 cells were lysed, immunoprecipitated with mAb C, electrotransferred to nitrocellulose membranes, and probed with mAb 452 and HRP-conjugated secondary antibody, as described in Materials and Methods. (c) Deficient binding of anti-CD13 antibodies to THP-1 cells transfected with siRNA for CD13 (L2 cells). U937 (left panel), THP-1 (center panel), or L2 cells (right panel) were incubated with the indicated mAb, washed, and incubated with a secondary FITC-labeled goat anti-mouse Ig antibody. (d) Inhibition of Aminopeptidase N enzymatic activity by anti-CD13 mAbs 452, WM-15, C, and E and a control IgG, and by the chemical inhibitor bestatin, in cell lines expressing human APN: U-937 cells, HEK cells transfected with human APN/CD13, and J774 murine cells expressing human APN/CD13. The graphs represent the arithmetic mean ± SD of three independent experiments in each cell line. ; .

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