Figure 11

Western blot analysis of cell signaling proteins, listed in methods. A498 or Caki-1 cells were treated either with 1 μM AEE788 or with 1 nM RAD001, or with a 1 μM AEE788-1nM RAD001 combination. Controls remained untreated. Drugs were applied for 24 h. Cells were then kept for 2 h in serum-free cell culture medium and subsequently stimulated for 30 min with EGF (100 ng/ml). Cell lysates were subjected to SDS-PAGE and blotted on the membrane incubated with the respective monoclonal antibodies. Beta-actin served as the internal control. The figure shows one representative from three separate experiments.