CALR mutants induce a specific activation of MPL. (A) Ba/F3 cells transduced to express Mock or CALRwt or mutants and MPL, EPOR, or G-CSFR were cultured for 10 days in the absence of any cytokine and their number was measured by a cell counter (Beckman Coulter). (B) Proliferation was assessed 72 hours after culturing Ba/F3-MPL cells expressing Mock, CALRwt, or the CALR mutants in the absence or in the presence of increasing doses of TPO (0.005, 0.015, 0.05, 0.15, 0.5, 1.5, and 5 ng/mL) by WST-1 proliferation assay. Dose-response curves are means (in percentage of viability of the maximum growth value) ± SEM (n = 3 in triplicate). Bonferroni multiple comparison test: *P < .05, **P < .001. (C) Ba/F3-MPL cells expressing different CALR constructs was examined by western blotting for the presence and phosphorylation status of various signaling molecules. Cells were serum- and cytokine-starved for 5 hours prior to 10 minutes stimulation with 10 ng/mL TPO. Expression of β-actin was used as loading control. (D) Immunoprecipitation (IP) using anti-FLAG antibody from lysates of Ba/F3 cells expressing a FLAG-tagged MPL and transduced with respective CALR constructs followed with western blotting.