Figure 3

Ets-1 activation by NO requires Ras signaling. Western blot of S-nitrosylated, active and total Ras in serum-starved MDA-MB-468 cells (a) transfected with NOS2 expression plasmid and treated with L-Arg or AG and (b) treated with EGF or DETANO. (c) Western blot of Ras-SNO and total Ras from MDA-MB-468 cells exposed to DETANO (0.5 mM) alone or in combination with N-acetyl cysteine (NAC) or sodium azide. (d) Ets-luciferase activity in MDA-MB-468 cells exposed to conditions as above. Significance to DETANO was determined by one-way ANOVA (**P < 0.01). (e) Western blot of Ras and Ets-1 (thr 38) phosphorylation in serum-starved MDA-MB-468 cells exposed to EGF or DETANO (0.5 mM) in the presence or absence of FTS. (f) Western blot of Ets-1 (thr 38) phosphorylation in serum-starved MDA-MB-468 cells exposed to EGF or DETANO (0.5 mM) in the presence or absence of Gö 6976. (g) Ets-luciferase activity in serum-starved MDA-MB-468 cells exposed to EGF or DETANO (0.5 mM) in the presence or absence of FTS or Gö 6976. Significance compared to control was determined by one-way ANOVA (*P < 0.05). (h) Schematic representing the NO-sensitive Ras/MEK/ERK/Ets-1 signaling pathway. AG, aminoguanidine; ANOVA, analysis of variance; DETANO, diethlylenetriamine NONOate; EGF, epidermal growth factor; ERK, extracellular signal-regulated protein kinase; Ets-1, erythroblastosis virus E26 oncogene homolog 1; FTS, farnesylthiosalicylic acid; L-Arg, L-arginine; MEK, mitogen-activated protein kinase; NOS2, nitric oxide synthase.