(A) BMMPCs that were not transduced (Unt) or were transduced with a retroviral vector expressing mCherry (mCh) or a vector encoding mCherry and one of two independent shRNAs specific for PLC-γ2 (shγ2-1 or shγ2-2) were lysed, and the abundances of PLC-γ2, pAkt, and pERK were measured by Western blotting analysis, with actin used as a loading control. (B) Effects of PMA and GM-CSF on Ras-GTP, pERK, and pAkt abundances in BMMPCs from WT and KrasG12D mice. BMMPCs from the indicated mice were treated with PMA or GM-CSF for the indicated times and lysed. Ras-GTP abundance was assessed by a Ras-RBD pull down assay and abundance of pAkt, and pERK was analyzed by Western blotting on the same lysates. Actin was used as a loading control. (C) BMMPCs were pretreated with PI-90 or U73122 (U73) and then were left unstimulated or were stimulated with GM-CSF or PMA for 15 min. Cells were then analyzed by Western blotting for the abundances of pAkt and pERK, with actin used as a loading control. (D) Analysis of CFU-GM growth from bone marrow cells from WT and KrasG12D mice that were cultured in a saturating concentration of GM-CSF (10 ng/ml) in the absence or presence of the inhibitors JakI, PI-90, U73122, or AKT VIII. Data from independent experiments corresponding the panels (B) and (C) are shown in figs S8 – S10. Data in (D) are mean values ± SD from three independent experiments.