Oncogenic H-ras activates 3 major pathways in erythroid progenitors. Freshly isolated fetal liver TER119^- cells were infected with bicistronic retroviruses encoding hCD4 alone (control vector) or oncogenic H-ras (H-ras.V12 mutant) and cultured overnight. The cells were starved for 2 hours and harvested. Levels of RalA-GTP, the active form of RalA, were analyzed by affinity purification of lysates with use of a GST fusion with RalA-binding domain of Ral BP immobilized on agarose beads. Phosphorylated p44/p42 ERK and Akt were measured by Western blotting (described in “Materials and methods”). For each tested signaling protein, the activated or phosphorylated form is shown in the top blot (indicated by a circled P) and the total protein on the bottom blot.