Low concentrations of H2O2 or nitrite induce Ras activation.

(A) Pb18 yeast cells were cultivated in modified YPD for 5 days at 37°C, subjected to a 24-h period of starvation with F12 medium and incubated with 0.1 mM H2O2 or 0.25 µM NO2 at pH 5.6 for different timepoints at 37°C. (B) Pb18 yeast cells were cultivated in modified YPD for 5 days at 37°C, subjected to a 24-h period of starvation with F12 medium and cultured with or without 250 µM FPT III inhibitor followed by treatment with 0.1 mM H2O2 or 0.25 µM NO2 at pH 5.6 for 1 h at 37°C. After yeast cells lysis (A) and (B), Ras activation was determined using the GST-RBD(Byr2) fusion protein, which binds with high affinity to the GTP-associated form of Ras. Ras-GTP (active) and total Ras (50 µg total protein) were assayed by western blots probed with anti-Ras antibody. Relative densitometric values of bands are shown in the bar graphs.