Effects of MAPK inhibitors on neural induction of ES cells. (A) Total cell lysates were collected from differentiating ES cells at indicated times under SFEB condition. Kinetic JNKs activation was analyzed by western blot. FGF1 dose-effect on differentiating ES cells was revealed by ERK phosphorylation at 30 min differentiation. (B) Downstream FGF signals were further detected with individual specific antibodies at 12 hr post-treatment of 40 ng/ml FGF1 (lane 3), or with inhibitors (lane 4) of PI3K/AKT (LY 294002, 10 μM), JNK1/2 (SP 600125, 10 μM), p38 MAPK (SB 203580, 20 μM), and ERK1/2 (U0126, 5 μM). After treatment with the inhibitors (C) or FGF1 (40 ng/ml) plus the inhibitors (D) from day 1 to day 3, the derived cells were collected for FACS analysis on day 6. The same concentrations of reagents were applied in these experiments. Representative results were shown from experiments done at least in triplicate.