Fig. 3

Identification of non-polysialilated CD56 as a novel ligand of DC-SIGN. a The presorbed anti-CD56 or isotype (Iso) control antibodies were incubated with PBL lysates. DC-SIGN-Fc (DC) binding to the captured CD56 alone or in the presence of inhibitors (EGTA and mannan, E-M) was assessed. ICAM-3-Fc (ICAM) was used as a negative control. b The binding of anti-Lewis sugars antibodies (Le) to the captured CD56 was assessed. Negative control (N.C.) was performed without anti-Lewis antibodies. a and b experiments were performed in triplicates and error bars represent ± SD of triplicates. c The immunoprecipitated DC-SIGN ligands (sDC) and CD56 (a-CD56) from activated PBLs, pretreated with biotin, were analyzed in immunoblot with streptavidin, left panel. The immunoprecipitated cell surface CD56 was analyzed in immunoblot with DC-SIGN-Fc and anti-CD56 antibodies, right panel. The arrow points out a common for the two immunoblots band. d Results of anion exchange (AEX) chromatography separation of immunoprecipitated CD56 from the activated PBL lysate. Fractions I and II were collected. e The fraction I (black bars) and fraction II (white bars) obtained in the AEX experiment were directly sorbed of ELISA plates and analyzed for binding anti-CD56 antibodies and DC-SIGN-Fc with or without of inhibiting mannan/EGTA (Man/EGTA) mixture