BLNK binding with JAK3 is essential for the inhibition of JAK3/STAT5 signaling, leading to cell-cycle arrest and apoptosis. (A) BKO84 cells were infected with a retroviral vector carrying BLNK. At 48 hours later, these cells were lysed and immunoprecipitated with αBLNK antibody or a control rabbit IgG (center panels), or with αJAK3 antibody or a control mouse IgG (right panels). These precipitates and the lysates (left panels) were analyzed by Western blotting with αJAK3 (top) or αBLNK (bottom) antibodies. (B) Tyrosine-to-phenylalanine mutants of BLNK. The numbers indicate the positions of tyrosine (Y) or substituted phenylalanine (F) residues in BLNK or BLNK mutants. Other single Y-to-F mutants, Y84F and Y119F, were not depicted. (C-E) BKO84 cells were infected with retroviral vectors carrying either BLNK or one of the BLNK mutants coupled via an IRES to rCD2. (C) Cells were lysed 48 hours after infection, immunoprecipitated with αBLNK antibody, and then blotted with αJAK3 (top) or αBLNK (middle) antibodies. The cell lysates were blotted with αJAK3 antibody (bottom). (D,E) At 72 and 96 hours after infection, rCD2⁺ cells were sorted by MACS. The sorted cells were lysed, and the lysates were analyzed by Western blotting with the indicated antibodies (D). Cell-cycle profiles of the sorted cells were analyzed as in Figure 2A (E). All data are representative of 2 independent experiments.