Fig. 5

RAS paralogs display different expression and activation patterns in undifferentiated vs. differentiated hiPSCs. a qPCR analysis of RAS-related genes in undifferentiated vs. differentiated hiPSCs. Results from three separate experiments, each carried out in triplicate, are shown as mean ± SD (ANOVA; *p < 0.05 and **p < 0.01). b Immunoblot analysis of RAS protein expression in undifferentiated (CM-100) and differentiated hiPSCs (CM-0 and non-CM). c The graph represents densitometric analysis of three independent experiments, each carried out in duplicates. All values were normalized to γ-tubulin and relative to CM-100. Data are shown as mean ± SD (ANOVA; **p < 0.01). d CRAF-RBD-derived pull-down of GTP-bound RAS in differentiated (CM-0 and non-CM) and undifferentiated (CM-100) hiPSCs were analyzed by immunoblotting of pan-RAS. γ-tubulin was used as loding control. e Immunoblot analysis of p-ERK1/2 showed that transient bFGF stimulation induces phosphorylation of ERK1/2 in KSR-starved hiPSCs after 15 min. The amounts of ERK1/2 and γ-tubulin were used as loading controls. f RAS paralogs were subjected to pull-down analysis in undifferentiated hiPSCs in the presence and absence of bFGF using two GST-fused RAS effectors, CRAF-RBD and PI3Kα-RBD. RAS-GTP pull down assays were followed by western blot analysis using pan-RAS antibody and RAS paralog specific antibodies. Immunoblots of total cell lysates were served as loading controls to detect pan-RAS, NRAS and KRAS. GST and pan-RAS were used as negative and loading controls, respectivley