Figure 4.
Increase in GTP-bound H-Ras in reactive astrocytes by redox stimulation. Acutely stimulated murine astrocytes were subjected to commercial c-Raf-1 RBD pull-down assay. Cells were grown and stimulated as indicated after or not pre-loading with the ROS scavenger N-Acetyl cysteine in complete serum (labels: SW, serum withdrawal; H2O2, hydrogen peroxide addition; NAC, N-Acetyl cysteine). H-Ras and K-Ras GTP-bound fractions were quantified by immunoblot after GST-RBD pull-down assay as described in Methods. Panel A shows immunoblot analysis of GTP-bound H-Ras and its flow-through (F.T.). The immunoblot is probed with the specific antibody anti-H-Ras (#MAB3291). Panel B shows immunoblot analysis of GTP-bound K-Ras and its flow-through (F.T.); the immunoblot is probed with the specific anti-K-Ras (Proteintech #12063-1-AP). Results represent the average of two independent experiments. Histogram representation of the quantitative densitometric analysis of GTP-bound fraction of each isoform and values are total Ras relative variation (see Supplementary Figure S14, Dataset Figure 4) **P < 0.0001; *P < 0.001; °P < 0.05 as compared with the untreated astrocytes (Student’s t-test).