The Ras pathway is activated in ovarian carcinoma cell lines and primary OSCs. (A) Ras-GTP and total Ras levels in the ovarian carcinoma cell lines were detected using a Ras activation assay kit as described in the Materials and Methods section. Representative data are shown. The blot was reprobed with antiactin as a loading control. HCT116, which does not express NF1, and HEY, which expresses mutant KRas (G12D), were used as a positive controls for Ras-GTP levels. Expression of Nf1 in each cell line [low or absent (-) versus readily detectable (+)] is indicated below each lane. (B) Immunohistochemical analysis of pMAPK in primary OSCs; representative examples are shown. OSC-32 (left panel) has a NF1 mutation and shows strong nuclear staining for pMAPK in the tumor cells, with absence of staining in the non-neoplastic stromal cells. No NF1 alterations were detected in OSC-33, which is negative for pMAPK expression (right panel).