Figure 6

N-Ras regulation of Stat1 through the Ras-ERK pathway. (a) N-Ras controls transcriptional activity of ISREs in fibroblasts. Relative luciferase activity of transfected reporter ISRE constructs versus their empty vector controls was measured as described in Materials and methods after 1 hour or 8 hours of serum stimulation in cultures of WT, N-ras^-/- or N-ras^-/- cells transfected with an appropriate N-Ras construct. The assays were carried out in triplicate, with error bars indicating standard deviation (***P < 0.001 versus WT; ⁺⁺⁺P < 0.001, ⁺⁺P < 0.01 versus N-ras^-/- fibroblasts). (b) Restored N-Ras expression in N-ras^-/- fibroblasts. Western immunoblot showing partial recovery of N-Ras expression after transfecting N-ras^-/- fibroblasts with a vector containing a single N-ras copy. (c) Regulation of Stat1 expression and activation through the ERK pathway. WT control fibroblasts were treated with different inhibitors as indicated and total Stat1 or pStat1 (Y701) levels were detected by immunoblot. Controls of activity of the kinase inhibitors are included in Figure 9e.