TpoR and CALR del52 interact directly to form stable complexes. (A) Coimmunoprecipitation analysis showed that TpoR and CALR del52 interaction is dependent on N-linker sugars of TpoR, especially N117-sugars and on the lectin binding domain of CALR. (B) Oligomerization analysis of CALR and TpoR by size-exclusion chromatography. Expression vectors coding for CALR del52 or extracellular domain (ECD) of TpoR and containing a metallothionein promoter were stably transfected (calcium phosphate) in S2 Drosophila cells with G418-containing medium. Expression of the proteins of interest was induced by copper treatment, and secreted forms of TpoR and CALR del52 were further collected in the supernatant. His-tagged ECD-TpoR was enriched by using a Nickel column. Finally, molecular-size distribution of CALR del52 and TpoR was measured by size-exclusion chromatography and fractions analyzed on MOPS 4% to 12% gel in the presence of DTT. (C) Glycosylation analysis with Endoglycosidase H and Peptide N-glycosidase F (N-glyc F or PNGase F) of the indicated fractions obtained from size-exclusion chromatography. (D) Cartoon representation of our Nano-BRET set-up. Because CALR del52 (del52) mutant interacts longer and in close proximity (<10 nm) to TpoR compared with the WT CALR, it enables BRET from the Nano-luciferase fused to the N-terminus of TpoR (NL) to the 618-ligand fluorophore (depicted by a red star) bound to the HaloTag (H) fused to the C-terminal tail of CALR. The BRET is symbolized by a shift in wavelength (460 nm [blue] to 618 nm [red]) of the bioluminescence emanating from the Nano-luciferase. (E) BRET between Nano-luciferase fused cytokine receptors and CALR fused to HaloTag using the Nano-BRET system. There is a strong energy transfer between TpoR and CALR del52 but not between EpoR and CALR del52. Black bars show standard deviation. ****P < .0001, unpaired nonparametric 2-tailed Student t test, 4 independent experiments each with 3 biological replicates.