FL binds bivalently to Flt3 ectodomain variants to form high-affinity complexes. (A-B) Isolation of Flt3_D1-D5:FL and Flt3_D1-D4:FL by SEC. Also shown are Coomassie-stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis strips corresponding to the peak fraction of the isolated complexes. (C) SEC on the Flt3_D1-D3:FL mixture at the end of an ITC experiment, showing that a large amount of Flt3_D1-D3 remains in the unbound form. Identical elution profiles were obtained in standard SEC experiments as well, in the presence of a large molar excess of FL. (D-F) Binding isotherms and thermodynamic parameters of FL binding to Flt3 ectodomains obtained by ITC. All ITC experiments were carried out by titrating recombinant human Flt3 ectodomain variants with FL.