Figure 4. The induced NOS (iNOS) pathway is activated in Alexander disease mouse models.

(a) Western blot shows increased expression of iNOS in GFAP^Tg mice (5 months old). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as a loading control. Quantification is shown in the right panel. *P=0.0042, t=5.857, df=4; unpaired t-test, n=3. Ctrl is age-matched wild-type littermates. (b) Quantification of nitrotyrosine protein levels reveals an increase in GFAP^Tg mice (5 months old). *P=0.0476, t=2.825, df=4; unpaired t-test, n=3. Ctrl is age-matched wild-type littermates. (c) Double-label immunofluorescence demonstrates increased expression of iNOS pathway markers: iNOS, nitrotyrosine (N-tyr), cyclic GMP (cGMP) and cyclic nucleotide-gated channel alpha 4 (CNGA4), in astrocytes of 3-month-old GFAP^R236H/+ and GFAP^Tg mice (arrows), but not in age-matched wild-type littermate controls (arrowheads). GFAP was used to identify astrocytes. 4,6-diamidino-2-phenylindole (DAPI) labels nuclei. Scale bar, 20 μm. See Supplementary Figs 8 and 9 for larger images.