H-rev107 inhibited activation of H-RAS induced by EGF. (A) HtTA cells plated in 10-cm dishes were transfected with H-rev107 or control vector for 24 h and then serum starved for 12 h. Cells were stimulated with EGF (50 ng/mL) for 5 min. Cell lysates were prepared, and the activated RAS (RAS-GTP) was isolated with RAF-RBD-conjugated agarose. The levels of total RAS, H-rev107 fusion proteins, and actin were measured by Western blotting. (B) HtTA cells were plated in triplicate, incubated overnight, transfected for 2 days with control vector, H-rev107 expression vector, and reporter plasmids, and were then stimulated with EGF (50 ng/mL) for 12 h at 37°C. Cell lysates were prepared and analyzed for the transactivation activity of ELK1 protein. Representative results from triplicate samples are expressed as mean ± SD. Student’s t-test: ***, P < 0.001.