Fig. 5. Dimerization of TpoR and of CALR del52 is mediated by the α-helical segment of CALR mutant C-terminus.

a Representation and sequence of CALR del52 C-terminal truncations used in proliferation assay. b Proliferation assay. Ba/F3 cells stably expressing TpoR in pMX-IRES-GFP vector were transduced with indicated CALR variants or an empty vector (pMSCV-IRES-mCherry) and sorted by FACS. 250,000 cells were washed and seeded in 10 mL of complete culture medium without cytokine and counted each day using an automated cell counter. Values represent mean of 3 independent experiments (±SD) (n = 3). Source data are provided as a Source data file. Data were analyzed using two-ways ANOVA followed by Sidak multiple comparison test at day 4 using the average of technical triplicate counting from 3 independent experiments (n = 3). c Cartoon representation of the crosslinking assay to assess homodimerization of TpoR in a productive orientation. o-PDM: ortho-phenylene dimaleimide. NEM: N-ethylmaleimide. Items in this figure were created with BioRender. d Crosslinking study of TpoR dimerization in presence of Tpo, CALR del52 full length or C-terminal truncations. Shown is a representative western blot (from 3 independent experiments) in denaturing and reducing conditions showing human TpoR monomers and o-PDM crosslinked dimers in the indicated conditions. Source data are provided as a Source data file. e Co-immunoprecipitation of CALR del52-HA full length by CALR del52-FLAG full length or truncated to assess dimerization. Shown are representative western blots in denaturing conditions (from 3 independent experiments). Source data are provided as a Source data file. f RosettaDock top scoring simulation of CALR del52 dimers. Structure of CALR del52 was modeled using AlphaFold 2.0¹⁸. CALR del52 monomers are shown in orange and pink. Residues of the CALR mutant C-terminus are shown in purple (Thr), dark blue (Arg) and red (Met). Close interactions (<3Ä) are shown by yellow dashed lines.