Figure 3.

Redox-stimulation decreased protein turnover of H-Ras. (A) Representative immunoblot analysis of Ras in response to serum withdrawal or hydrogen peroxide administration. Acutely stimulated murine astrocytes were lysed with RIPA and immunoblot analysis with commercial antibodies against total Ras, ERK 1/2 and phosphorylated (p-) ERK ½ is shown. Growing primary astrocytes (15 DIV in 10% FCS serum) were subjected to trophic deprivation (0% FCS serum) or hydrogen peroxide administration (H₂O₂ 500 μM in complete serum) in presence or not of cycloheximide (CHX) pre-treatment for the indicated times. β-actin bands served as a loading control. Densitometric analysis is reported below (See Supplementary Figure S9 Raw data from densitometric analysis of western blot relative to Figure 3 panel A) (B) Astrocytes deprived of serum (i) or hydrogen peroxide treated (ii) were subjected to immunoblot analysis with the indicated antibodies anti-H-Ras Millipore #MAB3291; anti-K-Ras Sigma R3400; anti-GFAP #MAB3402 Clone GA5. Tubulin bands served as a loading control. Densitometric analyses of the Western blotting experiments results are reported as histogram above each panel and performed using Image Studio Light version 5. (See Supplementary Figure S10 and S14 Raw data from densitometric analysis of western blot relative to Figure 3) **P < 0.0001; *P < 0.01; °P < 0.05 as compared with the untreated astrocytes (Student’s t-test).