Phlsec

For the recombinant human CLEC-2–6xHis tag and human podoplanin-rabbit Fc vector overexpression, the extracellular domain of the cDNA of human CLEC-2 and podoplanin were cloned into a pHLSEC (Addgene) and pFUSE (Invivogen) expression vectors. The constructs were used to transient transfection into human embryonic kidney HEK293T cells. Cells were grown in complete Dulbecco's modified eagle medium (DMEM; supplemented with 10% fetal bovine serum, glutamine 5%, and 100 U/mL of penicillin/streptomycin). Once 60% of cell confluence was achieved, the medium was exchanged for serum-free DMEM and transfected using the PEI method. Proteins secreted into the medium and were collected after 4 days. hCLEC-2–6xHis tag and h-podoplanin-rFc were purified using a Nickel-NTA Resin (Thermo Fisher) and protein A-coated beads (Thermo Fisher), respectively, in a gravity purification column. SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) was used to characterize proteins. Protein coding sequences of recombinant proteins can be found in the Supplementary Information.