Secretion and stability of plasma CALR-del52 from Calr-del52/WT KI mouse and patients with MPN. (A) Immunoprecipitation (IP) of murine plasma CALR-del52 and whole-blood lysates. Secretion rate of CALR-del52 was evaluated by comparing the cellular and plasmatic CALR-del52 levels from blood of KI-Calr^del52/WT mice expressing or not expressing TpoR. (B) Stability study of mutant CALR in plasma from patients with MPN harboring mutated CALR-del52. Samples (n = 8) from patients with MPN harboring mutated CALR-del52 maintained at 37°C for various time points were measured in duplicate by ELISA and analyzed using a 1-phase decay model (Prism6) to determine the averaged half-life (t½) and the coefficient of determination (R²). Error bars represent SDs. (C) Stability study of rhCALR-del52 in culture medium in absence of fetal bovine serum. A fixed amount of rhCALR-del52 was incubated in culture medium at 37°C for various lengths of time before measurement by ELISA analysis using a 1-phase decay model (Prism6) to determine the averaged half-life and R². Error bars represent SDs. (D) CALR mutant proteins after immunoprecipitation in various MPN patients (left) and CALR mutant proteins quantification by western blotting (optical density) of the same patients (right). Ctrl, control; mut, mutant.