Figure 1.

Oncogenic Ras isoforms differentially induce DNA synthesis in CV-1 cells. (a) CV-1 cells were cotransfected with HA-H-Ras^G12V and GFP-K-Ras^G12V expression vector or HA-H-Ras^G12V and GFP-N-Ras^G12N expression vector. Cells were fixed and subjected to immunostaining with anti-HA antibody. Representative images of single confocal section were presented. Scale bar, 10 μm. (b) Recombinant Ras proteins were purified as described in Materials and Methods. The purity of the purified Ras proteins was analyzed by SDS-PAGE and Coomassie blue staining. (c) Serum-starved CV-1 cells were microinjected with GST-Ras (0.5, 1 or 2 mg/ml) in combination with rabbit IgG. The cells were incubated with BrdU for 16 hr. DNA synthesis in the injected cells was determined by incubation with rat anti-BrdU antibody followed by TRITC-conjugated anti-rat antibody and FITC-conjugated anti-rabbit IgG antibody. The results were represented as means ± SD from three determinations with over 200 cells injected. (d) GTP binding activity of oncogenic H/K/N-Ras. GST-fusion Ras proteins (1.5 μg) were incubated with the varying concentrations of [³H]-GTP. Scatchard plot analysis was performed with the linear regression performed with Harvard graphics.