FIG. 1.

Stimulation of NF-κB-dependent transcription by the H-Ras(V12, C40) effector mutant. NIH 3T3 cells were transiently cotransfected with an NF-κB-responsive reporter (3x-κB-Luc, 0.5 μg) and with expression plasmids bearing the gene encoding H-Ras(V12), H-Ras effector mutants, or an empty vector control (VC) (1 μg each). Cell lysates were harvested 24 h posttransfection, and luciferase activity was assayed as described in Materials and Methods. Data are presented as multiples of the level of activation obtained for the vector control group, which was normalized to 1. Results are the means ± standard deviations of results of three independent experiments. (Gel) Total protein was isolated from a representative transfection experiment, and immunoblot analysis was performed for transgene expression. Protein samples (50 μg per lane) were resolved on a 10% polyacrylamide gel, transferred to nitrocellulose, and probed for hemagglutinin-tagged p21^Ras proteins with a hemagglutinin-specific antibody (BABCO, Berkeley, Calif.). Immunoblot assays for actin expression confirmed that relatively equal amounts of proteins were loaded in each lane.