Figure 5.

Cross talk between PI3K and ERK1/2 in response to HGF. (A) CaOV-3 cells plated on polyHEMA-coated dishes were pretreated with 50 µM PD98059 (PD), 200 nM wortmannin (Wt), or 20 nM rapamycin (Rp) for 30 minutes or (B) transfected with nonspecific (NS), ERK1/2 or Akt siRNA for 24 hours and then treated with HGF (10 ng/ml) for 15 minutes. Equal amounts of protein (20 µg) were analyzed by Western blot using specific antibodies recognizing phospho (p)-Akt, p-p70^S6K, or p-ERK1/2. The same membranes were stripped and reprobed with antibodies to total Akt, p70^S6K, and ERK1/2. β-Actin was also included as a loading control. The signal intensity was determined by densitometry and expressed as p-Akt, p-p70^S6K, and p-ERK1/2 relative to total Akt, p70^S6K, ERK1/2, and β-actin for each sample. (C) Cell apoptosis was determined with the TUNEL assay. The number of apoptotic cells was counted, and the bar diagram summarized the percentage of apoptotic cells from triplicate determination. Error bars indicate the SD of the mean. *P < .05 versus untreated controls.