Effects of expressing wild-type and mutant SHP-2 proteins on ERK and Akt activation and survival in Ba/F3 cells. (A) Duplicate aliquots of Ba/F3 cells transduced with retroviruses encoding various SHP-2 constructs (wild-type [WT], D61Y, or E76) were collected after 4 days of growth and selection in medium containing IL-3 and puromycin. Parental Ba/F3 cells are labeled P. The cells were starved for 5 hours and lysed without stimulation (–) or after exposure to 10 ng/mL IL-3 for 10 minutes (+). The bottom panel shows SHP-2 expression, which was equivalent in cells transduced with the WT, D61Y, or E76K vectors and elevated above the levels in parental Ba/F3 cells or in cells infected with the empty vector (not shown). Ba/F3 cells expressing all of the SHP-2 constructs showed low levels of phosphorylated ERK (p-ERK) and Akt (p-Akt) after starvation, with robust and equivalent activation in response to IL-3. Parental and transduced Ba/F3 expressed total levels of ERK2 and Akt. (B) Ba/F3 cell counts after IL-3 withdrawal. Ba/F3 cells infected with the MSCV-puro vector (▪), and cells expressing either WT SHP-2 (♦), the D61Y SHP-2 mutant protein (▵), or the E76K SHP-2 mutant protein (○) were plated in triplicate at 1 × 10⁶ cells/plate in the absence of IL-3. Cells were counted starting on day 8.