JAK2^ex13InDel activates signaling through βc-associated cytokine receptors. (A-C) HEK293 cells expressing βc were transfected with the α chains of the IL-3R, IL-5R, or CSF2R (GM-CSF receptor), together with a luciferase-based STAT5 reporter (Spi_Luc) and JAK2^WT, JAK2^V617F, or JAK2^ex13InDel. STAT5 transcriptional activity was measured in the presence or absence of the respective cytokines at 10 ng/mL (n = 3). Data were analyzed with a 2-way analysis of variance and Tukey’s correction for multiple comparisons (*P < .05, **P < .01, ***P < .001, ****P < .0001). (D) The FERM domain mutation Y114A was introduced into JAK2^ex13InDel. Ba/F3 cells expressing JAK2^{Y114A/ex13InDel} cells were cultured with or without IL-3. JAK2 double-mutant cells did not proliferate upon IL-3 withdrawal (n = 3). (E) Parental and Ba/F3 cells expressing JAK2^WT, JAK2^V617F, and JAK2^ex13InDel grown with IL-3 supplementation were washed to remove IL-3 and replated with or without IL-3. Cells were harvested after 4 hours. βc immunoprecipitates were resolved on SDS-PAGE and incubated with a phosphotyrosine antibody (top). A representative experiment out of 3 independent repeats is shown. βc immunoprecipitates were incubated with a JAK2 antibody (bottom). A representative experiment out of 3 independent repeats is shown. WCL, whole-cell lysate.