Absence of confounding cross-talk signaling in cells transformed by gag-akt/RafCAAX.

(A) Cell lysates derived from IL-3–deprived H7 parental cells, from H7 BCR-ABL.A54 cells in the absence of IL-3, and H7gag-akt/RafCAAX cells, also in the absence of IL-3, were subjected to SDS-PAGE and antiphosphotyrosine immunoblotting (right panel indicates whole-cell lysate) or, additionally, aliquots of lysates were subjected to immunoprecipitation with anti-Shc and then processed for SDS-PAGE and antiphosphotyrosine immunoblotting (left side). Afterward, the blot was stripped and reprobed with anti-Shc. Asterisk (*) indicates putative SHIP band, whereas (**) indicates Shc. (B) Aliquots of the aforementioned cell lysates derived after IL-3 starvation in the presence of 1% FCS or 10% FCS were immunoprecipitated with anti-Cbl, then immunoblotted with antiphosphotyrosine. Top arrow (*) demonstrates pp120 cbl; bottom arrow (**) demonstrates putative ppShc. Next, the blot was stripped and then reprobed with anti-Cbl. (C) The aforementioned cell lines were starved of IL-3 or had IL-3 (WEHI-3 CM) added for 1 hour, and then cell lysates were made and were either used as whole-cell extracts (right panel) or were immunoprecipitated with antiphosphotyrosine. The precipitates and the whole-cell lysates were subjected to SDS-PAGE and immunoblotting for Stat5α. There was no increase by IL-3 treatment of tyrosine-phosphorylated Stat5α in cells with p210BCR-ABL. In H7gag-akt/RafCAAX1 cells, tyrosine phosphorylation of Stat5 was seen at this time point (60 minutes). For H7 parental cells, a shorter time course (5-30 minutes) of IL-3 treatment (not shown) revealed tyrosine phosphorylation of Stat5α.