Fig. 5. BMP4 induces phosphorylation of SMAD1/5, while VEGF induces phosphorylation of Erk1/2 during EB differentiation. (A) ES cells were differentiated up to day 1.5 (left) or day 2.75 (right) in serum-free conditions in the presence of either bFGF or BMP4. (B) EBs harvested from day 1.5 differentiated in serum-free conditions were treated with either bFGF or BMP4 for 30 and 60 minutes. (C) ES cells were differentiated in serum-free conditions with BMP4. Three days after, BMP4 was washed off overnight and EBs were treated with VEGF for 30 minutes. EB cell lysates were subjected to SDS-PAGE, followed by immunoblotting with pSMAD1/5, SMAD1 (A,B), pERK1/2, ERK1/2, pAKT1/2/3 or AKT1/2/3 (C). One out of three experiments is shown.