Fig. 10. The effects of APC on GTP-loading and the protein level of Ras. (A) DLD-1 cells were transfected with 0.5 µg of either pCMV or pCMV-APC with or without 0.1 µg of pMT3-RAS-L61. Cells were harvested 72 hours after transfection. RAS pull-down analysis was performed to detect GTP-loaded active RAS (De-Rooij and Bos, 1997) as described in Materials and Methods. Western blot analyses were performed on whole-cell lysates to detect p-ERK, APC, Pan-RAS and -tubulin. The GTP-loaded Pan-RAS proteins (RAS-GTP) were also detected by western blot analysis using anti-Pan-RAS antibody. (B) DLD-1, SW480 or NIH 3T3 cells were transfected with 0.5 µg of pCMV or pCMV-APC. Cells were harvested 72 hours after transfection, and p-ERK, APC, Pan-RAS and -tubulin were detected in whole-cell lysates by western blotting. RAS pull-down analysis and detection of RAS-GTP were performed as described in A.