BLNK-inhibition of JAK3/STAT5 signaling pathway is Btk independent, but induction of p27kip1 protein and cell-cycle arrest are Btk dependent in BKO84 cells. (A-C) BKO84, si-Luc BKO84, and si-Btk BKO84 cells were infected with a retroviral vector carrying BLNK coupled via an IRES to rCD2, and rCD2⁺ cells were sorted by MACS 72 and 96 hours after the infection and used for each analysis. (A) Cell-cycle profiles of the BLNK-transduced si-Luc BKO84 and si-Btk BKO84 cells were analyzed as in Figure 2A. (B) Cell lysates were analyzed by Western blotting with the indicated antibodies. (C) Semiquantitative RT-PCR analysis of p27^kip1 mRNA and GAPDH mRNA (cDNA quantity control) in BLNK-transduced si-Btk BKO84 and si-Luc BKO84 cells 72 hours after infection. All data are representative of 2 independent experiments.