Constitutive activation of JAK3/STAT5 signaling by autocrine IL-7 expression confers proliferation ability on BLNK^−/− pre-B leukemia cells. (A) Activation state of JAK3, STAT5a/b, and Erk1/2 in primary BLNK^−/− pre-B leukemia cells (top panel) and of STAT5a/b in BLNK-pBLs (bottom panel) was assessed by Western blotting of the cell lysates with antibodies against an activation-coupled phosphorylated site in each protein. The same filters were reblotted with the antibodies against each protein. BM indicates pre–B cells purified from bone marrow cells of BLNK^−/− mice that did not develop the leukemia. LN1–3 indicates pre-B leukemia cells purified from enlarged lymph nodes of 3 individual BLNK^−/− mice. (B) BKO84 cells were transduced with either SOCS1 or SOCS1 F59D via retrovirus vectors containing EGFP as a selection marker. EGFP fluorescence in the infected (open) and nontreated (shaded) cells at the indicated times after infection was analyzed by flow cytometry. The percentage of EGFP⁺ cells in each infected sample is indicated. (C) RT-PCR analysis of IL-7 mRNA in primary BLNK^−/− pre-B leukemia cells (left panel; the same samples as in panel A), BLNK-pBLs, A-MuLV–transformed pre–B-cell line 18-81, B-cell lymphoma line WEHI231, and IL-7–producing stromal cell lines ST2 and OP9 (right panel). HPRT mRNA was amplified to control cDNA quantity. (D) Growth curves of BLNK-pBLs and 18-81 cultured in the medium containing αIL-7R (■), class-matched control antibody (▵), or none (○) are shown. Each symbol represents mean numbers (± SD) of triplicate cultures. All data except panel A (top) are representative of 3 independent experiments.