Journal: PLoS ONE

Article Title: Trans-Activation between EphA and FGFR Regulates Self-Renewal and Differentiation of Mouse Embryonic Neural Stem/Progenitor Cells via Differential Activation of FRS2α

doi: 10.1371/journal.pone.0128826

Figure Lengend Snippet: (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.

Article Snippet: Ras and Rap1 activity in the mouse embryonic NSPCs in response to ephrin-A1 and FGF2 were measured using a Ras activation assay kit and Rap1 activation assay kit (Cell Biolabs, Inc., San Diego, CA, USA; Cat. #STA-400 and #STA-406-1), respectively.

Techniques: Phospho-proteomics, Western Blot, Concentration Assay, Activation Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: Trans-Activation between EphA and FGFR Regulates Self-Renewal and Differentiation of Mouse Embryonic Neural Stem/Progenitor Cells via Differential Activation of FRS2α

doi: 10.1371/journal.pone.0128826

Figure Lengend Snippet: (A) FGF2-induced activation of FGFR leads to phosphorylation of both Grb2 and Shp2 binding sites on FRS2α. Both the Shp2- and Grb2-mediated pathways were found to lead to activation of MAP kinase via activation of the Ras pathway. Rap1 was not significantly activated. (B) Co-stimulation with FGF2 and ephrin-A1 induced strong and sustained activation of FRS2α. Both the Ras- and Rap1-mediated pathways resulted in quick and robust MAP kinase activation, augmenting self-renewal of NSPCs. (C) Ephrin-A1-induced activation of EphA led to delayed weak activation of MAP kinase, and appeared to be mediated mainly by the Shp2-Ras and Shp2-Rap1 pathways via trans-phosphorylation of FGFR by EphA. Activation of Ras through the EphA-mediated Grb2-Ras pathway was weak. Dotted lines and grey letters indicate decreased signals compared with solid lines and black letters. Two FRS2α molecules are included in the complex to conveniently separate the signal transduction pathways mediated by ephrin/EphA4 and FGF/FGFR. The binding stoichiometry has yet to be studied.

Article Snippet: Ras and Rap1 activity in the mouse embryonic NSPCs in response to ephrin-A1 and FGF2 were measured using a Ras activation assay kit and Rap1 activation assay kit (Cell Biolabs, Inc., San Diego, CA, USA; Cat. #STA-400 and #STA-406-1), respectively.

Techniques: Activation Assay, Phospho-proteomics, Binding Assay, Transduction