Identification of the epitope for E4G10. (A) Alanine scanning was performed in full-length mVE-cadherin cDNA expression plasmids, with substitutions at amino acid positions 1-15. Mock-transfected, as well as 293 cells expressing murine wild-type (mWT) and mutant mVE-cadherin expression plasmids were stained with E4G10 followed by goat anti–rat-PE secondary antibody to determine relative MFI by flow cytometry. The mAb-binding activity on the alanine-substituted VE-cadherin is measured relative to binding activity on mWT VE-cadherin and normalized for transfection efficiency (percent relative MFI, see “Materials and methods”). Bar graphs represent the average of 3 separate experiments with SDs. (B) Diagram of mWT and mN-cadherin_1-10/mVE-cadherin_11-739 (1-10 N/VE) and mN-cadherin_1-15/mVE-cadherin_16-739 (1-15 N/VE) chimeric molecules. VEC indicates VE-cadherin. (C) Results of E4G10 binding to the 1-10 N/VE and 1-15 N/VE chimeric molecules compared to mWT and mock transfected (n = 3).