Analysis of Jak-Stat signaling in BM and spleen of JAK2-Ex12 transgenic mice. (A) Western blot analysis of indicated total and phospho proteins in BM lysates from WT and MxCre;Ex12 mice. Bar graphs represent the average ratio of phospho and total proteins for signal intensity of respective proteins (n = 3 per each genotype). (B) Phosphorylation levels of Stat5 protein at Tyr705 detected in nuclei of BM cells (sternum) and spleen cells by immunohistochemistry (IHC) staining. The histograms show the percentages of nuclei positive for phospho-Stat5 counted using the Aperio ImageScope image analysis software (left). The group sizes were: n = 10 for WT and MxCre;Ex12 mice, and n = 6 for MxCre;V617F mice. The values represent the mean ± standard deviation. Intracellular phospho-flow analysis of phospho-Stat5 levels in BM and spleen from indicated mice (middle) (n = 6 per genotype). Relative levels of phospho-Stat5 protein measured in spleen cell lysates using the MSD (right). (C) Phosphorylation levels of Stat3 protein at Tyr705 detected in nuclei of BM cells (sternum) and spleen cells by immunohistochemistry staining. The histograms show the percentages of nuclei positive for phospho-Stat3 (left). The group sizes were: n = 10 for WT and MxCre;Ex12 mice, and n = 6 for MxCre;V617F mice. Intracellular phospho-flow analysis of phospho-Stat3 levels in BM and spleen from indicated mice (middle). Relative levels of phospho-Stat3 protein measured in spleen cell lysates using the MSD technology (right). The values represent mean ± SEM (n = 3 per group). Intracellular phospho-flow analysis of (D) phospho-Stat1, (E) phospho-Erk1/2, and (F) phospho-Akt1 levels in BM and spleen from indicated mice. Values on Y-axis indicate the MFI of indicated proteins (n = 6 mice per genotype). One-way ANOVA with subsequent Bonferroni post-test was used. *P < .05; **P < .01; ***P < .001. (G) Blood counts, survival, and spleen weight of lethally irradiated recipient mice (n = 4 per group) transplanted with 1 × 10⁶ BM cells from non-induced donor mice with the indicated genotypes. Tamoxifen was injected 6 weeks after transplantation, as indicated by the blue arrows. (H) Analysis of BM (top) and spleen (bottom) cells of transplanted mice euthanized 24 weeks after transplantation. MFI, mean fluorescence intensity.