Enhanced STAT and ERK signaling in the 2-oncogene compared with the 1-oncogene model. (A) Western blot analysis of pSTAT1, pSTAT3, pSTAT5, pERK1/2, pAKT, SHIP, and BCL-2 proteins in the indicated cell lines. Cells were starved for 16 hours in 0.5% FBS medium and then stimulated with starve media or with saturating concentrations (*) of mIL-3 (50 ng/mL), hIL-6 (50 ng/mL), mSCF (50 ng/mL), or GM-CSF (50 ng/mL) for 5 minutes at 37°C. Total-cell lysates were separated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride, and probed with indicated antibodies or GAPDH as a loading control. (B) Western blot analysis of pSTAT and pERK1/2 proteins in MN1+CTL, MN1+HOXA9, and HOXA9+CTL cells pretreated as described for panel A. (C-D) Densitometry analysis of Western blot analysis of phosphorylated STAT and ERK1/2 proteins in the indicated cell lines stimulated with IL-3/IL-6/SCF (C) or GM-CSF (D; normalized to GAPDH, mean ± SD, n = 2 [see panel A and data not shown]).