MR mediates recognition and internalization of omega-1 by human DCs. (A) Human moDCs were incubated for 1 h with PF-647–labeled recombinant WT omega-1, the glycosylation mutant, or the RNase mutant and analyzed for uptake of antigens by FACS analysis. One representative experiment with duplicate samples out of two experiments is shown. Bars represent mean ± SD. (B) Human moDCs were incubated for 1 h with PF-647–labeled omega-1 and, where indicated, either preincubated (pre) with EGTA to prevent omega-1 binding to CLRs a priori or treated afterward with EGTA (post) to remove any CLR-bound omega-1 from the cell surface. One of two independent experiments is shown, and data represent mean ± SD of duplicates. (C and D) A binding/internalization assay of natural (C) and recombinant omega-1 (D) by immature moDCs was performed analogous to B after preincubation with the indicated reagents. Binding and internalization are shown relative to control pretreatment. (C) Data are based on five experiments and are shown as mean ± SD. (D) One of two independent experiments is shown, and data represent mean ± SD of duplicates. (E and F) 3T3 cell line–expressing MR (E) and K-SIGN–expressing DC-SIGN (F) or parental control cell lines (3T3 and K-562) were incubated with PF-647–labeled omega-1 and SEA in the presence or absence of EGTA to determine specificity. One representative experiment based on duplicate samples out of two is shown. Bars represent mean ± SD. * and #, P < 0.05; ** and ##, P < 0.01; ***, P < 0.001 for significant differences compared with control conditions (*) or between test conditions (#) based on two-sided Student’s t test. H58F, RNase mutant; MFI, mean fluorescence intensity; N71/176Q, glycosylation mutant.