Figure 1.

Reactive oxygen species (ROS) selectively induce K-Ras and H-Ras protein levels in primary astrocytes. The direct effects of hydrogen peroxide addition in regulating the protein levels of K-Ras and H-Ras in cultured astrocytes were measured by western blot analysis. The kinetics of total p21^Ras expression and K-Ras isoform in cultured astrocytes subjected to H₂O₂ treatment is shown in (Panel A). The growth medium (high-serum 20%) was switched into a low-serum (2%) medium containing or not H₂O₂ and western blot analyses were performed on Radioimmunoprecipitation (RIPA) buffer extracts. Total p21^Ras protein shows constitutive low levels in untreated astrocytes and increases upon oxidative stimulation (Panel D). K-Ras (sc-521) levels increased in 5 min and rapidly decreased, reaching the basal levels at 60 min of stimulation (Panel C). Conversely, H-Ras protein levels peaked at 60 min and remained high for up to 120 min (Panel B); representative immunoblots with antibodies against H-Ras (sc-520) and pan-Ras (H259) show the same immune-reactivity pattern with differences in sensitivity. Monoclonal anti-β actin was used as loading control. Experiments were carried out in triplicates and statistical significance obtained by Student’s t-test. * p < 0.01 as compared with the untreated normal astrocytes (Student’s unpaired test); ° p < 0.02 as compared with the untreated sample (Student’s matched pairs test).