FIG. 2.

Ras expression in stably transfected WRT cells. Total cell lysates were prepared and protein determinations were made. (A) Western blot probed with antihemagglutinin antibody to detect transfected Ras. Samples from left to right are vector-transfected (V) cells, Ras^G37 (pool 1), Ras^S35 (pool 10b) (2 μg each), and Ras^V12 (clone 3, 10 μg). The levels of Ras expression were similar in multiple pools and single-cell clones of cells transfected with Ras^S35 and Ras^G37. Ras^V12 was expressed at levels approximately 7.5- to 10-fold lower than those of the effector mutants. (B) Western blot probed with pan-Ras antibody to detect cellular Ras. Samples from left to right are WRT (100 μg), vector-transfected (V) cells, Ras^G37 (5 μg), and Ras^V12 (50 μg). Densitometric analysis revealed that Ras^V12 was overexpressed 4-fold and Ras^G37 was overexpressed 40-fold relative to cellular Ras. Cellular Ras expression was not altered by transfection of Ras^V12 or the mutants.