Western blot analysis of the elements in GM-CSF signal transduction pathway in PtenΔ/Δ; Nf1^+/− mice induced at PND8 and in control littermates at age 3 weeks (2 weeks post–poly[I:C]). After serum starvation (S) and stimulation with GM-CSF (G) or IL-3 (I), mouse BM cells were lysed in a density of 2 × 10⁷/mL in 2× Laemmli Sample Buffer (Bio-Rad Laboratories). One aliquot with 20 μL per well was loaded onto a 4% to 20% Mini-PROTEAN TGX gel (Bio-Rad). (A) Comparison of the activities of Akt and MAPK pathways in mice with WT and PtenΔ/Δ; Nf1^+/− induced at PND8 in response to stimulation by GM-CSF and IL-3 at a physical concentration. Representative data from 8 experiments in littermate mice (age 3-4 weeks) are shown. (B) Comparison the activities of Akt and MAPK pathways in mice with PtenΔ/Δ with or without Nf1^+/− in response to stimulation by GM-CSF. Representative data from 3 experiments are shown. (C) Comparison the activation of CREB and expression of Egr-1 in mice with WT and PtenΔ/Δ; Nf1^+/−. Representative data from 4 experiments are shown. CREB and Egr-1 were detected on 2 separate blots. p, phosphorylated protein; t, total protein. Additional relevant semiquantitative data are presented in supplemental Figure 7.