Figure 3

In vitro molecular responses to gefitinib, RAD001 and the gefitinib and RAD001 combination. SKBR3, JIMT-1 and MCF7-HER2 cells were treated for 72 h with indicated concentrations of gefitinib (Gef), RAD001 (RAD) or the combination of both drugs at a 200:1 (Gef:RAD) molar ratio. Cell lysates were prepared from harvested cells and expression of phospho- and corresponding total protein were analyzed with Western blotting. (A) Representative images of protein bands. (B) The expression of phosphorylated proteins, as shown in (A) was measured by densitometry and normalized to the vehicle control expressed as 1 and denoted by a dotted line in each histogram. β-actin was used to control for equal loading. The MDA-MB-468 cell line with a high EGFR and negligible HER2 expression was used to control for specificity of the anti P-EGFR and P-HER2 antibodies.