CALR mutants activate and rescue cell surface expression of mutated TpoR harboring a retention signal in its C-terminal domain. STAT5 transcriptional activity in γ2A cells of CALR mutants when coexpressed with TpoR WT (A), TpoR RT1 (B), and TpoR RT2 (C). Values shown represent the average of 3 independent experiments each done with 3 biological repeats ± standard error of the mean. (D) Autonomous growth at 72 hours, as determined by CTG assays of Ba/F3 cells expressing the indicated CALR and TpoR WT or RT2. Cells were infected with retroviruses coding for WT or mutant CALR and WT TpoR or TpoR RT2. Similar expression was determined by flow cytometry for markers downstream of the IRES of the retroviral vectors (mCherry for CALR and GFP for TpoR variants). Shown are means ± standard error of the mean. Kruskal-Wallis test with Steel posttest at 5% significance level. ***P < .001. Flow cytometry analysis of cell surface expression of the TpoR WT (E), TpoR RT1 (F), and TpoR RT2 (G) coexpressed or not with CALR WT or CALR del52. ICD, intracellular domain; RT, retention signal.