Figure 5

Dose-dependent reduction of K-Ras GTP in MIA PaCa-2 (A) and immortalized K-Ras12D expressing cells (B). Exponentially growing MIA PaCa-2 cells serum-starved starved for 9 h in DMEM with 0.1% FBS and then treated for 18 h with 0 (DMSO control), 1, 3 or 10 μM of TLN-4601. At the end of the treatment, cells were stimulated with EGF (100 mg/ml) for 5 min. HPNE-KRAS cells were grown in normal serum condition and treated for 18 h with 10 μM of TLN-4601. Pull-down analyses and western blot analyses with K-Ras-specific antibody were done to determine the level of activated and total K-Ras protein expression. Data are representative of one to three independent experiments. Vinculin was used as a loading control.