N-Ras regulation of Bax and PERP expression. (a) Transcriptional activation of Bax is N-Ras-dependent. Relative luciferase activity of transfected Bax construct versus their empty vector controls was measured in WT, N-ras-/- and N-ras-/- with partially restored N-ras expression cells as described in Materials and methods. The assays were carried out twice, each time in triplicate, with error bars indicating standard deviation (**P < 0.01; ***P < 0.001 versus control; +P < 0.05; ++P < 0.01 versus N-ras-/- fibroblasts). (b) Transcriptional activation of Perp is N-Ras-dependent. Relative luciferase activity was measured in transfected WT, N-ras-/- and H-ras-/-/N-ras-/- fibroblasts as well as in double knockout fibroblasts with partially restored expression of either H-ras or N-ras. The assays were done twice, each time in triplicate, with error bars indicating standard deviation (*P < 0.05; **P < 0.01; ***P < 0.001 versus WT cell lines; +P < 0.05; ++P < 0.01 versus N-ras-/- fibroblasts; Ψ P < 0.05; ΨΨ P < 0.01 versus H-ras-/-/N-ras-/- fibroblasts). (c) Western immunoblot showing recovery of H-Ras and N-Ras expression after transfection of double knockout H-ras-/-/N-ras-/- fibroblasts with vectors containing a copy of either H-ras or N-ras. (d) Regulation of Bax expression and activation through the ERK and p38 pathways. Control fibroblasts were treated with different Ras-effector inhibitors as indicated in Materials and methods, and total Bax levels were detected by immunoblot. (e) Controls of activity of the chemical inhibitors on their corresponding molecular targets.
Castellano et al. Genome Biology 2009 10:R123 doi:10.1186/gb-2009-10-11-r123