Figure 4.
Specific activation of Raf or PI3-kinase is not sufficient to promote anchorage-independent growth of human cells. (A) Expression of ΔRaf1-22W or p110-CAAX was confirmed in the described HEK–HT cells by immunoblotting with an anti-Raf1 antibody or by RT–PCR with primers specific for p110. Actin and GAPDH levels serve as loading controls. These cells also were infected with an empty vector. (B) Specific activation of either the MAP-kinase or PI3-kinase pathway by ΔRaf1-22W or p110-CAAX, respectively, was confirmed in the described HEK–HT by immunoblotting with antibodies specific for the phosphorylated forms of ERK1 and ERK2 or Akt. Total ERK 1/2 and Akt levels serve as a loading control. (C) Anchorage-independent growth of HEK–HT cells expressing ΔRaf1-22W or p110-CAAX, as calculated from the average number of colonies observed from three plates, and expressed as the percent of colonies observed in Ras12V-transformed cells. A total of 50,000 HEK–HT cells expressing H-Ras12V seeded in soft agar yielded 311 ± 46 colonies.