All bcr/abl mutants confer factor-independent growth to FL5.12 cells.
(A) IL-3–independent growth of FL5.12 cells expressing the indicated bcr/abl proteins. FL5.12 cells were transduced with the indicated viral supernatant. Following transduction, FL5.12 cells were grown for 40 hours and FACS analysis was performed to determine the percentage of GFP+ cells. At 48 hours after transduction, cells were deprived of WEHI-conditioned medium (source of IL-3) and maintained in IL-3–free media for an additional 64 hours at which time aliquots were prepared for FACS and protein analysis. Vector control cells (MigR1) died without IL-3. (B) Immunoblots of whole cell lysates from panel A. For comparison, lysates were prepared from parental FL5.12 cells either growing in 10% WEHI-conditioned medium (lane 2) or deprived of IL-3 for 12 hours (lane 1). For each lane, 106 cells were lysed in a final volume of 100 μL lysis buffer and 12 μL of each lysate was loaded on SDS-PAGE (6% gel for 24-11 blotting and 9% gel for PY20 blotting). The immunoblotting antibodies and size markers (kd) are indicated. In the top 2 panels the bcr/abl proteins are indicated by a ■.
