Figure 4. Modulation of EGFR activity during the first hours of myoblast differentiation.

All results were normalized to the result obtained for GM conditions. A. Myoblasts were lysed in proliferation (GM) and at different times in differentiation conditions (DM). Western blot analysis of EGFR on whole cells extracts. EGFR expression decreases by 64% after 24 h in DM (n = 5). B. Myoblasts were fixed in proliferation (GM) and at different times in differentiation conditions (DM). Flow cytometry was performed on non-permeabilized myoblasts. For the control condition myoblasts were only incubated with the secondary antibody. A significant decrease of EGFR at the plasma membrane is observed after 9 h in DM (n = 5). To control the specificity of the antibody against EGFR, we verified that the antibody did not bind any more to myoblasts transfected with a siEGFR. C-D-E. Total EGFR, phospho-p42/p44 MAPK and Myogenin expressions were assessed by Western blot. α-Tubulin expression was used as a loading control. C. Efficiency of EGFR inhibitors. Myoblasts were cultured in GM for 1 h with AG1478 at 10 µM or PD153035 at 3 µM. Western blot analysis shows a significant decrease of phospho-p42/p44 MAPK expression but no difference of EGFR expression (all n = 3). D. Myogenin expression after 24 h treatment with EGFR inhibitors in proliferating conditions (n = 4). E. Phospho-p42/p44 MAPK and EGFR expression during the first hour of differentiation (n = 5).