Lentivirus-mediated gene silencing in transduced TonB cells. (A) Dot blots of RFP fluorescence and side scatter (SSC) from TonB cells transduced with dcH1-GL4-SR, dcH1-b3a2-SR, dcH1-237SHP2-SR, dcH1-STAT5-SR, and dcH1-Gab2-SR 4 days after lentiviral transduction in the presence of IL-3. (B) For immunoblotting, TonB cells transduced with control dcH1-GL4-SR (lane 2), dcH1-121gfp-SR (lane3), dcH1-b3a2_1-SR (lane 4), or with anti-STAT5 shRNA, anti-Gab2, or anti-SHP2 shRNA (lane 5 in each blot), respectively, were lysed 4 days after transduction. Lane 1 shows untransduced TonB cells. The immunoblots were probed with anti-SHP2, anti-Gab2, anti-STAT5, anti-STAT3, and anti-Gab1 antibodies as indicated, and reprobed with anti-ERK2 antibody as loading control. (C) Number of trypan blue-negative TonB cells was determined starting 4 days after lentiviral transduction with different shRNAs (set day 0 on x-axis, average from 3 independent experiments) in the presence of IL-3. Triangles depict transduction with dcH1-GL4-SR or mock-transduced cells (control), and the almost identical cell number at day 14 was set 100%. Circles show cell numbers of TonB cells transduced with viruses encoding dcH1-b3a2_1-SR, dcH1-237SHP2-SR, dcH1-Gab2-SR, and dcH1-STAT5-SR as indicated. In a different set of experiments anti-GFP shRNAs were included as an additional control with almost identical results as shown. (D) Number of trypan blue-negative TonB cells was determined in the presence of doxycycline to induce BCR-ABL expression and in the absence of IL-3 (average from 3 independent experiments). Labeling as in panel C. (E) Number of trypan blue-negative TonB cells from cultures shown in panel D after readdition of IL-3 after 4 days (set day 0 on x-axis, averages from 3 experiments). Labeling as in panel C.