Loss of growth factor gene expression and reduced tyrosine phosphorylation of BCR-ABL and STAT5 in cultured BCR-ABL–transduced IL-3^−/− cells.

(A) RT-PCR analysis of RNA isolated from representative clones generated from IL-3^−/− and +/+ cells transduced with BCR-ABL only (lanes 1-5) or BCR-ABL and IL-3 (lanes 6 and 7). Lanes 1 and 2 show results for 2 BCR-ABL–transduced IL-3^−/−clones; lanes 3-5 for 3 BCR-ABL–transduced +/+ clones; and lanes 6-7 for 2 clones of IL-3^−/− cells cotransduced by IL-3 and BCR-ABL. Lane 8 was a negative control (water control). (B) Western blot analyses of cell lysates from control (MIG-transduced) cells with or without IL-3 (lanes 1 and 2); 4 clones of BCR-ABL–transduced IL-3^−/− cells (lanes 3-6); and 3 clones of BCR-ABL–transduced +/+ cells (lanes 7-9). The cells were incubated in the presence or absence of IL-3 (as indicated below each lane) for 16 hours and lysates from equal numbers of cells separated on 8% polyacrylamide gradient gels. Filters were first probed with antiphosphotyrosine (4G10) and anti–phospho-STAT5 (P-STAT 5A/B) antibodies, then stripped and reprobed with anti–ABL antibodies and anti-STAT5 (STAT5A). The positions of prestained molecular weight markers are indicated on the left side of each blot. (C) Western blot analysis of IL-3–transduced IL-3^−/− (lanes 1 and 2) and +/+ cells (lanes 3 and 4); BCR-ABL– and IL-3–transduced IL-3^−/− cells (lanes 5 and 6); and BCR-ABL–transduced +/+ cells (lanes 7 and 8). Culture conditions and antibodies were the same as in panel B.