Characterization of the EMR2-ligand interaction. (A) The EMR2-ligand interaction is Ca²⁺ dependent. (B) Preincubation with anti–EGF-like domain 4 antibodies (1B5 and 2B1) results in the ablation of the binding of multivalent EMR2 probes to CHO-K1. An anti–EGF-like domain 1 antibody (CLB-CD97/1) and a control Ab (N418) had no effect on binding. (C) FACS analysis showing that binding of CHO-K1 cells by the largest isoform of human CD97 is similar to that of EMR2. (D) Generation of stably transfected CHO-K1 cells expressing transmembrane EMR2/1-5 and EMR2/1,2,5 proteins (left panel). Expression of cell-surface EMR2 was determined by flow cytometric analysis using 2A1 mAb and an isotype-control Ab (data not shown). The formation of cell rosettes in single-cell suspensions (right panel). EMR2/1-5–expressing cells (white bar) formed more cell rosettes than did EMR2/1,2,5–expressing cells (gray bar) and pcDNA3.1-transfected cells (black bar) (n = 20). Error bars are equal to one standard deviation.