Figure 1.

RAS Activity Is Induced in Early Differentiation

(A) Real-time PCR analysis showing the relative expression of markers of pluripotency (Nanog and Oct4) and markers of the three germ layers (Brach, Tbx5, FoxA2, Gata4, K18 and Nestin) at the indicated days of embryoid body (EB) differentiation, compared with undifferentiated cells.

(B) Differentiated cells (EBs) were lysed at the indicated days. RAS-GTP pull-down assay followed by western blot analysis of active pan-RAS (RAS-GTP) or western blot analysis of OCT4 or AKT (protein loading) is shown.

(C) Western blot analysis of OCT4 or AKT (protein loading) at the indicated time points following LIF removal.

(D) Quantification of expression of OCT4 and SSEA1 proteins (data shown in Figure S1) at the indicated time points following LIF removal. Quantification was performed using a Nikon NIS-Element D, as detailed in the Experimental Procedures.

(E) RAS-GTP pull-down assay was followed by western blot analysis using pan-RAS antibody (RAS-GTP) or using the indicated RAS isoform-specific antibodies (H-RAS-GTP, K-RAS-GTP, or N-RAS-GTP). The total expression of pan-RAS (RAS) or of each isoform (H-RAS, K-RAS, or N-RAS) was examined by western blot.

(B, C, and E) Values represent densitometry analysis of at least three independent experiments. Data shown are mean ± SD from three independent experiments. ^∗p < 0.05 statistically significant by Student's t test.