Raf-1 is not a downstream effector in the signal transduction pathway used to signal VEGF induction in Ha-ras–transformed cells (NIH3T3R). (A) NIH3T3R cells were stably transfected with a mutant inhibitory allele of Ras [Ras(N17)] or Raf-1 [Raf301] before transient transfection with 5 μg of the 385-bp VEGF reporter plasmid and exposure to hypoxia. Values represent at least five independent transfections. Error bars represent 1 SD of the mean. (B) Transient transfections with Raf301 (0.1, 1, and 10 μg) and 385-bp reporter construct (2.5 μg) and exposure to hypoxia. (C and D) The effect of MAPKs on signaling VEGF induction by hypoxia. Kinase defective alleles of ERK1 and ERK2 were cotransfected with VEGF reporter constructs to examine their effect on inhibiting VEGF induction by low-oxygen conditions. The relative fold induction refers to the ratio of luciferase activity measured in treated cells relative to the activity observed in the untreated controls. Values represent the means of at least three independent transfections. Error bars represent 1 SD of the mean.