(A) Representative flow plots of healthy preleukemic and leukemic p53^–/–Lnk^–/– BM cells stained with of anti-B220 and intracellular IgM (μ–heavy chain, ICμ) antibodies after fixation. (B) Combined B220 and intracellular ICμ staining divide B cells into subpopulations at differentiation stages, which correlate with distinct IL-7 responsiveness. BM cells were starved, stimulated with or without IL-7, and subsequently fixed and stained as described in A, along with anti-pSTAT5 antibodies. Representative histograms of pSTAT5 signal for each population are shown. Dotted vertical lines indicate IL-7 control levels. Percentages of cells responded to IL-7 are indicated. P values are calculated from 2-tailed Student’s t test when compared with WT cells stimulated by IL-7. (C and D) The dot plots show the percentages of pSTAT5⁺ cells (C) and the MFI of pSTAT5⁺ signals (D). (E) Lnk^–/–p53^–/– leukemic blasts show elevated basal activation of pSTAT5. Representative phosphoflow shows the activation status of basal-level pSTAT5 in BM B220^loICμ^– cells in comparison with leukemic cells after 1 hour of starvation. The dot plots show the quantification of relative MFI of phosphoflow signals. *P < 0.05; ***P < 0.001; ns, not significant, 2-tailed Students’ t test. n = 4–10 mice per group in all above experiments. (F) Lnk^–/–p53^–/– leukemic blasts were starved and stimulated with or without IL-7 for 20 minuets. Cell lysates were subjected to WB analysis with indicated antibodies to phospho- or total JAKs. (G) HEK293T cells were transfected with mock or JAK3, along with either human or mouse Flag-tagged LNK expression constructs. Cell lysates were either precipitated with anti-Flag antibodies or directly and subjected to WB analysis with indicated antibodies. (H) BaF3/IL-7R cells were stably infected with retroviruses expressing either vector alone, or WT or R384E LNK mutant. Cell growth in the presence of IL-7 was quantified daily and graphed.