Cell-surface ligand-binding analysis. (A) A schematic representation of the biotinylated mouse Fc fusion proteins. The EGF-like modules are represented as triangles, gray circles represent the mouse Fc region, and the small black circles indicate the biotinylation signal. (B) Western blot analysis of EMR2-mouse Fc fusion proteins. An Fc-specific antimouse immunoglobulin G (IgG) horseradish peroxidase (HRP) was used to detect the purified mouse Fc fusion proteins: EMR2/1,2 mouse Fc (lane 1), EMR2/1,2,5 mouse Fc (lane 2), EMR2/1,2,3,5 mouse Fc (lane 3), and EMR2/1,2,3,4,5 mouse Fc (lane 4). Lanes 5 to 8 were loaded with the same EMR2 mouse Fc proteins after in vitro biotinylation and probed with Extravidin-HRP. (C) FACS profile showing that CHO-K1 cells only interact with fluorescent beads coated with the largest EMR2 isoform, but not with other isoforms. (D) The ligand binding is protease sensitive, demonstrating the requirement of cell-surface proteins for the EMR2-ligand interaction.