Figure 9.

NΔ87L R-Ras and Q87L R-Ras activate PI3K in 3T3 cells. NIH 3T3 cells were transfected with empty pCMV vector (lanes 1 and 2), pCGN NΔ87L R-Ras (lanes 3 and 4) or pCGN Q87L (lanes 5 and 6). Cells were treated with 0.37% DMSO (odd lanes) or 25 μM LY294002 (even lanes) for 30 min and lysed. Whole cell lysates were subjected to SDS-PAGE and phosphorylation of Akt on serine 473 was detected with a polyclonal antibody. Phosphorylated Akt was used as a reporter of PI3K activation. The phospho-Akt blot was stripped and reprobed for HA-tagged R-Ras using the 12CA5 mAb. Total Akt was detected with a distinct polyclonal antibody to assess protein loading.