Fig. 3

ETV2 increases FLK1⁺ cells through the miR-126/MAPK pathway. A, B iFLAG-ETV2 mESCs were differentiated with ± Dox for 3.5 days and subjected to western blot analysis for SPRED1 (A) and phosphorylated ERK1/2 (B). β-ACTIN and ERK1/2 were used as loading controls. (A’, B’) The relative protein expression of SPRED1 and p-ERK1/2 was normalized against β-ACTIN and total ERK1/2, respectively. Differentiated Etv2^−/− mESCs were also included for the analysis. n = 3, *p < 0.05, **p < 0.01. (C) iFLAG-ETV2 mESCs were differentiated for 4 days in serum-free (upper) or for 3.5 days in serum (lower) conditions in the presence or absence of Dox. The resulting cells were analyzed for FLK1 expression by flow cytometry. U0126 (5 μM) or U0124 (5 μM) was treated during the differentiation. C’ Quantification. n = 3, *p < 0.05. D Differentiated iFLAG-ETV2, iFLAG-ETV2-MAP2K1-8E (iFLAG-ETV2-M) or iFLAG-ETV2-SPRED1 (iFLAG-ETV2-S) mESC ± Dox were analyzed for FLK1 expression by flow cytometry. D’ Quantification. n = 3, *p < 0.05