Fig. 6.

Abi1 knockdown impeded in vivo competitive expansion of p185^wt cells. (A) Expression of Abi1 in Ba/F3 cells, p185^wt cells transduced with non-silencing shRNA (p185^wt control) or Abi1 shRNA (p185^wt Abi1 Ri), as well as the cells rescued from bone marrow (BM), peripheral blood (PB) and spleen of the diseased mice injected with p185^wt cells transduced with either non-silencing shRNA or Abi1 shRNA. The 100 μg total proteins from these cells were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and western blotted (WB) with indicated antibodies. Molecular markers are indicated. The arrow indicates p185^Bcr-Abl. (B) In vivo competitive expansion of p185^wt GFP cells and p185^wt Abi1 shRNA cells. The p185^wt cells were transduced with MSCV-based retroviruses expressing either GFP or Abi1 shRNA. The GFP-positive p185^wt cells were sorted by fluorescence-activated cell sorting to a purity of >95%. The GFP-positive cells then were mixed with p185^wt cells expressing Abi1 shRNA at a ratio of 1:1. The mixed cells were either cultured in vitro for 5 weeks or injected into Balb/c mice (1 × 10⁶ cells per mouse) through tail vein. The cells derived from p185^wt cells were rescued from bone marrow and peripheral blood (data not shown) of the diseased mice by selection with puromycin. The rescued cells and the cells cultured in vitro were subjected to flow cytometry analysis.