Fig. 5. KRAS^G12D activates the RAF1/MEK/ERK1/2 and PI3K/AKT pathways in granulosa cells. (A) RAS activity in wild-type ovaries during ovulation, as measured by a GST pull-down assay using RAF1 RAS-binding domain (RBD) as the bait. NT, non-treated. (B) RAS-GTP levels increased in immature LSL-Kras^G12D;Amhr2-Cre and LSL-Kras^G12D;Cyp19-Cre ovaries, as measured by the GST pull-down assay using both RAF1 RBD and p110 RBD. (C) Phosphorylation of MEK1/2, ERK1/2 and AKT in wild-type and LSL-Kras^G12D;Cyp19-Cre ovaries after eCG/hCG treatment. Total ERK1/2 and AKT are shown as loading controls. (D-F) Localization of phospho-ERK1/2 in ovaries. The level of phospho-ERK1/2 was low in immature wild-type mouse ovaries (D), but was increased in the large antral follicles 2 hours after hCG injection (E). By contrast, the phospho-ERK1/2 level remained low in LSL-Kras^G12D;Cyp19-Cre ovaries after the same treatment (F). (G-I) Immunofluorescence of phospho-AKT in wild-type ovaries. (G) Immature ovary; (H) 48 hours after eCG; (I) 2 hours after hCG. (J-L) Immunofluorescence of phospho-AKT in LSL-Kras^G12D;Cyp19-Cre ovaries. (J) Immature ovary before eCG treatment (abnormal follicle-like structures indicated by arrows); (K) 48 hours after eCG treatment; (L) 2 hours after hCG treatment. Three to six ovaries from different animals of each genotype were analyzed in each of these experiments.