Dissection of the EMR2 ligand. (A) FACS analysis of CHO cell mutants shows that EMR2 binds a CS-bearing proteoglycan. PgsB-618 lacks all GAG side chains, PgsD-677 lacks heparan sulfate side chains, and Lec1 cells lack N-linked carbohydrates. (B) Cell attachment assays using BSA (□), fibronectin (▧), EMR2/1-5 mouse Fc (⊞), and EMR2/1,2 mouse Fc (▪). Each point represents the mean ± SEM of triplicate wells. The experiments were performed 3 times with similar results. (C) Enzymatic digestion of GAG side chains show that EMR2 binds to a CS-B– or CS-C–bearing proteoglycan. (D) FACS analysis of CHO-K1 cells treated with 10 mM sodium chlorate or 10 mM sodium chlorate and 10 mM magnesium sulfate. (E) The EMR2-ligand interaction is blocked by exogenous GAGs. Efficient blocking was observed when incubated with CS-B (♦) and CS-C (▵), but not HS (▪) or CS-A (□). Note that the blocking effect is dose dependent. The figure is a representative of 3 independent experiments with similar results.