IFN-γ disrupts the low-affinity binding interaction between TPO and c-MPL. (A) Evaluation of the TPO:c-MPL binding affinity by MST in the presence (red) or absence (black) of IFN-γ. Without IFN-γ, the TPO:c-MPL interaction was characterized by a high-affinity (left panel) and a low-affinity (right panel) binding site. Addition of IFN-γ resulted in specific disruption of the low-affinity site in a dose-dependent manner. (B) Evaluation of the TPO:c-MPL binding affinity by MST in the presence of 100-fold molar excess concentration of hematopoietic cytokines (SCF and Flt3L). The TPO:c-MPL interaction was unaffected by SCF or Flt3L. (C) Evaluation of the binding affinity between IFN-γ and c-MPL by MST. IFN-γ did not interact with c-MPL. Data are mean ± SEM; curve fit by nonlinear regression. The K_D for each condition is listed in Table 1.