Characterization of EGFR in the MM cell lines. Levels of mRNA were revealed by quantitative PCR (A), EGFR RNA quantity indicates EGFR gene expression vs GAPDH. The standard deviation expresses the result of three different quantizations and expression of EGFR protein was revealed by quantitative PCR. The western blot (B) shows the levels of EGFR and phosphorylated EGFR protein (P-EGFR) in MM cell lines. Cell extracts (100 μg) were probed for phospho-specific EGFR p-Tyr-PY20 and detected with ECL. Blots were stripped and then re-probed for EGFR. Actin was used as loading control. C, effect of EGF on phosphorylation of EGFR, AKT and ERK. The cells were cultured in complete medium for 24 and then treated with EGF (100 ng/mL) for 15 and 30 min. Western blot of total lysates (60 μg) indicates that the addition of EGF at a concentration of 100 ng/mL significantly increased EGFR phosphorylation already after 15 minutes. AKT also become phosphorylated in Ist-Mes-1 and MPP89, whereas ERK become phosphorylated in MPP89.