Binding of soluble mutant CALR proteins to TpoR at the cell surface. (A) Cartoon representation of our exogenous CALR, cell coculture NanoBRET setup. HaloTag (H) is fused to the C-terminal of CALR, nano-luciferase (NL) is fused to the N-terminal of TpoR, and 618-ligand is a fluorescent molecule with very high affinity for HaloTag. The circle represents a bioluminescence resonance energy transfer (BRET) phenomenon that occurs only when the energy donor (NL) is within 10 nm of the energy acceptor (618-ligand). (B) BRET detection between CALR-HaloTag and cell-surface nano-luciferase-TpoR in a stable cell coculture assay. HEK293 cells stably expressing either CALR WT or del52-HaloTag were cocultivated overnight in presence of 618-ligand with HEK293 cells stably expressing NL-TpoR. Data from 3 independent experiments were pooled and values were normalized to the NL-TpoR and CALR WT-Halo condition. Statistical analysis (Jmp pro14) was performed by a 2-tailed student t test and aforementioned P values. (C) Cartoon representation of our assay to measure binding of rhCALR-del52 to BaF3 TpoR Calr^WT and BaF3 TpoR Calr^mut cells. Cells were incubated for 15 minutes with varying amounts of rhCALR-del52 before extensive washing. (D) Western blotting showing the presence of rhCALR-del52 bound to BaF3 TpoR Calr^WT and BaF3 TpoR Calr^mut cells after 15 minutes incubation with different concentration of rhCALR-del52.