BLNK negatively regulates the JAK3/STAT5 signaling pathway in pre-B leukemia cell lines and primary pre–B cells. (A-C) BKO84 cells were infected with retroviral vectors carrying either BLNK cDNA or no cDNA insert (MOCK) coupled via an IRES to rCD2. rCD2⁺ cells were sorted by MACS 48, 72, and 96 hours after infection and used for each analysis. (A) Cell-cycle analysis was done as in Figure 2A. (B) Cell lysates were analyzed by Western blot analysis with the indicated antibodies. (C) Surface expression of IL-7Rα and pre-BCR in rCD2⁻ cells (solid line) and rCD2⁺ cells (dotted line) were analyzed by flow cytometry. Nonstained controls are shown in gray. (D) κ⁻ λ⁻ B220⁺ pro/pre–B cells were purified from the bone marrow of WT or BLNK^−/− mice by MACS sorting, cultured in the medium containing IL-7 for 96 hours, lysed, and then analyzed by Western blot analysis with the indicated antibodies. All data are representative of 3 independent experiments. The reason for the increased phospho-ERK1/2 in both mock- and BLNK-introduced cells (panel B; see also Figure 6B) is unclear, but it might be due to the stresses that the cells might have suffered during the retroviral infection. (E,F) Parental, si-Luc, or si-p27^kip1 BKO84 cells were transduced with BLNK and rCD2⁺ cells were sorted at the indicated time points as in panels A to C, and analyzed by Western blotting (E) or for cell-cycle profiles (F), as in panels B or A, respectively.