Podoplanin is expressed on 293T cells and binds to CLEC-2. (A) 293T cells were either control transfected with empty vector or transfected with a podoplanin expression construct. Cells were stained with anti-podoplanin antibody 18H5 and analyzed by flow cytometry (black filled area: control transfected cells stained with isotype antibody, grey filled area: control transfected cells stained with 18H5, grey line: cells transfected with podoplanin expression plasmid and stained with 18H5). The results of a representative experiment are shown on the left side, the average of four independent experiments is presented at the right side. Error bars indicate SEM. (B) The experiment was carried out as described for (A), but binding of soluble CLEC-2 to podoplanin or control transfected cells and to CEM×174 cells was analyzed. The results represent the average ± SEM of the GMCF measured in three (CEM×174) and four (293T, 293T-PDPN) independent experiments. (C) 293 T-REx CLEC-2 cells were doxycycline treated to induce CLEC-2 expression or PBS treated, and binding of soluble podoplanin-Fc or Fc-control protein was analyzed. The results represent the average ± SEM of the GMCF measured in three independent experiments. Dox: doxycycline, GMCF: geometric mean channel fluorescence, PDPN: podoplanin, SEM: standard error of the mean.