Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage
Fig 3
Identification of epitope of the G8 mAb.
An alanine-scan library of BAI1 was constructed. The G8 and reference 5D6 mAbs were screened for binding to each individual BAI1 variant. A. Identification of candidate critical clones for G8 mAb binding was determined in duplicate by high throughput flow cytometry. For each point, background fluorescence was subtracted from the raw data that were then normalized to G8 reactivity with wild type BAI1. For each mutant clone, the mean binding value was plotted as a function of expression represented by control antibody reactivity. Candidate critical clones (red circles) were identified with a threshold (dashed line) of <40% binding of the G8 mAb and >50% of binding of the control 5D6 mAb to wild type BAI1. B. Identification of validated critical residues for G8 mAb binding. Candidate critical residues for G8 were rescreened in quadruplicate and the mean binding reactivities obtained. Validated critical residues for G8 binding (outlined in red) were residues whose mutations that resulted in <20% of wild type binding but positive (>70% binding) for binding of the 5D6 control mAb. Additional validated secondary residues (outlined in blue) were identified that did not meet the threshold guidelines but whose decreased binding activity and proximity to critical residues suggested that they may be part of the antibody epitope. C. Visualization of validated critical residues for G8 mAb binding. Critical residues (red spheres) and secondary residues (blue spheres) which may also contribute to binding were visualized on two Phyre-generated model structures that were combined to model BAI1 residues 262–934. The G8 critical residues were visualized on a model of the BAI1 thrombospondin repeat domains based on the structure of properdin (PDB ID# 1WOR; [15].