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

<p>All results were normalized to the result obtained for GM conditions. <b>A</b>. 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>B</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. <b>C-D-E</b>. Total EGFR, phospho-p42/p44 MAPK and Myogenin expressions were assessed by Western blot. α-Tubulin expression was used as a loading control. <b>C.</b> 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). <b>D</b>. Myogenin expression after 24 h treatment with EGFR inhibitors in proliferating conditions (n = 4). <b>E</b>. Phospho-p42/p44 MAPK and EGFR expression during the first hour of differentiation (n = 5).