Research Paper Volume 6, Issue 8 pp 602—620

Figure 3. hESC-produced factors act on multiple biochemical pathways.

Western immunoblotting analysis with GAPDH or β-actin as loading controls, from human and mouse myoblasts serum starved for one hour then treated for 20 minutes with 50% differentiation medium and 50% specified medium, with or without a MEKi (10 μM) to analyze crosstalk with MAPK pathway. (A) Immunoblot of the Notch pathway, specifically Delta-1 (dll1) and active Notch-1 (Nicd1), (B) Quantification of Dll1 and Nicd1 expression in human myoblasts. The relative expression level was normalized by GAPDH and presented as the expression level relative to that of human myoblasts treated with just differentiation medium. Significant differences were identified by Student's t tests (*p<0.002 and **p<0.05). Error bars indicate standard error of the mean (n=3). (C) Western immunobloting analysis of BMP pathway proteins pSmad 1/5/8 with β-actin as a loading control, as in (A). (D) Quantification of pSmad1/5/8 expression in human myoblasts. The relative expression level was normalized by βactin and presented as the expression level relative to that of human myoblasts treated with just differentiation medium. Significant differences were identified by Student's t tests (*p<0.05). Error bars indicate standard error of the mean (n=3). (E) Western immunobloting analysis of BMP pathway protein pSmad 1/5/8 with βactin as a loading control, from human and mouse myoblasts treated for 24 hours with 50% differentiation medium and 50% specified medium +/− MEKi (10 μM).