Fig 5. Ligand-specific activation of FRS2α, MAP kinase and Akt in mouse embryonic NSPCs.

(A-D) Time course tyrosine phosphorylation of MAPK (ERK) and Akt in response to ephrin-A1 (0.5 μg/ml) and FGF2 (20 ng/ml), respectively, in mouse embryonic NSPCs. Cells were starved in growth factor-free medium containing 0.5% BSA and 20 mM HEPES buffer for 4 h. Activated phospho-MAPK (p-MAPK) and total amounts of MAPK (total MAPK) were detected by immunoblotting with anti-phospho-p44/42 MAPK (Thr202/Tyr204) and anti-p44/42 MAPK antibodies, respectively, using 20 μg of cell lysate for each time point. Phospho-Akt (p-Akt) and the total amount of Akt (total Akt) were also detected by immunoblotting with anti-phospho-Akt and anti-Akt antibodies, respectively. SU5402 (final concentration of 25 μM) was added 3 h before exposure to the ligand. Activation of MAP kinase and Akt were quantified in four different experiments (bottom: representative blotting result) and values were expressed as the ratio of p-MAPK/total MAPK or p-Akt/ total Akt. Bars represent the SD. * p<0.005, ** p<0.001, *** p<0.0001 (n = 4) compared to the value at the 0 time point in each treatment using the two-tailed Student’s t test. (E, F) Time course of Ras and Rap1 activation in response to ephrin-A1 (0.5 μg/ml) and FGF2 (20 ng/ml), respectively, in mouse embryonic NSPCs. Experiments were carried out twice with similar results. One of the blots is shown. Mean values of active (GTP-bound) and total Ras and Rap1 under various conditions were evaluated with the ratio of GTP-bound Ras/total Ras and GTP-bound Rap1/total Rap1, respectively.