FIG. 6.

2G12 and DC-SIGN binding sites on gp120 are nonoverlapping. (A) 2G12 can bind to gp120-Fc prebound to DC-SIGN. gp120-Fc was used to stain BC7-DC-SIGN⁺ cells. After a washing step, 2G12 was added for 20 min, followed by another washing, and a PE-conjugated monoclonal anti-kappa antibody was used to detect 2G12 binding. Note that 2G12 alone in the absence of gp120-Fc did not show any nonspecific binding. In addition, no positive staining was detected by using gp120-Fc mutants lacking 2G12's epitope. (B) Wild-type gp120-Fc and representative gp120-Fc mutants lacking 2G12's epitope were used to stain BC7-DC-SIGN⁺ cells. PE-conjugated goat anti-human Fc antibodies were used to detect gp120-Fc binding to DC-SIGN. There was no significant difference in DC-SIGN binding between these glycosylation mutants and wild-type gp120-Fc.