Figure 6.

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.