Fig. 4. Involvement of the EGFR in G3EGF functions. (A) Cell lysate was prepared from cultures before and after serum withdrawal and immunoprecipitated with anti-EGFR antibody, followed by western blotting probed with anti-phosphotyrosine antibody. U87 cells expressing G3EGF had lower levels of tyrosine-phosphorylated EGFR (pEGFR) after serum withdrawal, but EGFR expression was similar at different time points. Densitometry readings of protein bands are provided below the blots. (B) Both types of cells were starved and then treated with EGF (100 ng/ml) in the presence or absence of AG1478. EGFR phosphorylation was abolished by AG1478 in both types of cells, whereas the protein levels of EGFR were not affected. (C) Cultured in DMEM containing 1% FBS in the presence or absence of AG1478, the growth-inhibitory effect of AG1478 on vector-transfected cells was much more severe than on G3EGF-transfected cells (AG1478– vs AG1478+: **P<0.001; *P<0.05). (D) When AG1478 was added to the cultures after serum withdrawal, cell apoptosis was accelerated more obviously in the vector-transfected cells (from 13% to 45% apoptosis, or a 250% increase) than in G3EGF-transfected cells (from 39% to 52% apoptosis, or a 33% increase). All the experiments were repeated three times.