Myelin decrease in the absence of R-Ras1 and/or R-Ras2. A, Coronal sections from adult (P90) mouse brains stained with DAPI showing a remarkable reduction in the thickness of the corpus callosum in the double mutant compared with the control. Lower-magnification images of the corpus callosum are shown in the boxed inset (interaural 4.40–4.76 mm and bregma 0.61–0.97 mm; Paxinos and Franklin, 2013). B, Quantification of corpus callosum thickness in these coronal sections showing a decrease in R-Ras1^−/−;R-Ras2^−/− compared with control. Corpus callosum thickness was analyzed in several positions: position 0 in x-axis means a measurement in middle line (R-Ras1^−/−;R-Ras2^−/ 169.7 ± 16.5 μm, control 428.6 ± 43.1 μm, ***p < 0.0001); position 0.5 is a measurement in cingulate cortex (R-Ras1^−/−;R-Ras2^−/− 158.7 ± 7.8 μm, control 321.3 ± 40.7 μm, ***p = 0.0002 in right, R-Ras1^−/−;R-Ras2^−/− 155.1 ± 15.8 μm, control 342.4 ± 30.2 μm, ***p < 0.0001 in left); position 1 is a measurement in secondary motor cortex (R-Ras1^−/−;R-Ras2^−/− 184.3 ± 18.2 μm, control 365.9 ± 59.5 μm, **p = 0.0011 in right, R-Ras1^−/−;R-Ras2^−/− 181.8 ± 29.9 μm, control 408.2 ± 41.6 μm, ***p < 0.0001 in left); position 1.5 is a measurement in primary motor cortex (R-Ras1^−/−;R-Ras2^−/− 158.9 ± 12.3 μm, control 305.9 ± 66.8 μm, **p = 0.005 in right, R-Ras1^−/−;R-Ras2^−/− 166.7 ± 31.2 μm, control 352.5 ± 30.3 μm, ***p < 0.0001 in left). C, D Dual staining of longitudinal sections of adult (P90) corpus callosum (C) and optic nerves (D) with anti-MBP and anti-NF200 (red) reveals myelin deficits in R-Ras1^−/−;R-Ras2^−/− mice relative to controls. E, Immunoblot of corpus callosum lysates from P90 double-mutant mice compared with controls also shows this reduction in MAG and MBP. Western blot of adult (P90) optic nerve lysates confirmed a significant decrease in the myelin marker protein expression like MAG and MBP in single and double mutants compared with controls (n = 3). F, Significant decrease in MAG and MBP expression in the corpus callosum. Bar graph represents mean ± SD of the change relative to the control of measurements normalized to GAPDH from three different experiments: MAG R-Ras1^−/−;R-Ras2^−/− 72.48 ± 15.91% (**p = 0.004); MBP R-Ras1^−/−;R-Ras2^−/− 44.52 ± 20.53% (***p < 0.0001). G, Significant decrease in MAG and MBP expression in the optic nerve. Bar graph represents mean ± SD of the change relative to the control of measurements normalized to GAPDH from three different experiments: MAG R-Ras1^−/− 72.89 ± 13.29% (**p = 0.0083), R-Ras2^−/− 63.65 ± 9.45% (**p = 0.0026), R-Ras1^−/−;R-Ras2^−/− 52.82 ± 13.19% (**p = 0.0035); MBP R-Ras1^−/− 69.24 ± 18.41% (**p = 0.0057), R-Ras2^−/− 56.36 ± 20.54% (**p = 0.0014), R-Ras1^−/−;R-Ras2^−/− 43.47 ± 19.03% (***p = 0.00016). n = 3. Scale bars: A, 500 μm; C, D, 5 μm.