Binding of podoplanin to CLEC-2 requires adequate podoplanin glycosylation and is independent of divalent ions. (A) The indicated CLEC-2 mutants were transiently expressed on 293T cells and expression (white bars) and binding of podoplanin-Fc (black bars) analyzed by flow cytometry. The results represent the average ± SEM of the GMCF measured in three independent experiments. (B) The Fc-control protein or the podoplanin-Fc fusion protein was transiently expressed in the indicated CHO cell lines. CHO Lec1 cells are defective in N-acetylglucosaminyltransferase (no complex N-glycans are generated), CHO Lec2 cells lack the CMP-sialic acid transporter (no sialylated glycoconjugates are generated). The supernatants of the transfected cells were harvested, concentrated and analyzed by Western blot, using the podoplanin-specific D2-40 antibody  (top panel) or a Fc-specific antibody (bottom panel). (C) The proteins generated in (B, control Fc-protein was 2-fold diluted) were incubated with CLEC-2 expressing 293 T-REx cells and bound protein was detected by FACS. The results represent the average ± SEM of the GMCF measured in three independent experiments. (D) Expression of DC-SIGN and CLEC-2 was induced on 293 T-REx cells by doxycycline treatment and the cells incubated with ZEBOV-GP-Fc or podoplanin-Fc, respectively, in the presence of PBS (dark bars) or 2 mM EDTA containing FACS buffer (white bars). Bound proteins were detected by flow cytometry. The results represent the average ± SEM of the GMCF measured in three independent experiments. GMCF: geometric mean channel fluorescence, PDPN: podoplanin, SEM: standard deviation of the mean.