Fig. 1: Retinal injury induces Nogo-A protein release in the mouse vitreous.
a–c Compared with intact condition (no treatment) and PBS treatment, intravitreal injection of NMDA induced the loss of retinal ganglion cells expressing Nogo-A and the specific cell marker RBPMS. In contrast, glial expression of Nogo-A did not change in glutamine synthetase (GS)-expressing Müller cells after NMDA-induced injury. Three mice per condition were examined. d By Western Blotting, the level of Nogo-A protein did not significantly change in retinal lysates treated with increasing doses of NMDA (0–40 nmoles) although P.Stat3 and P.Erk1/2 were upregulated in a dose-dependent manner in response to retinal damage. e Western Blot analysis of mouse vitreous (4 μL/well) revealed that NMDA induced the increase of Nogo-A and TNFα proteins in a dose-dependent manner. For quantitative analysis, 3 mice were used per condition. With Rb173A antibody which recognizes the Nogo-A specific domain encoded by exon 3, 4 proteins were observed at ~80 kDa (Nogo-A-F1), ~68 kDa (Nogo-A-F2), and at ~200–250 kDa (Full-length Nogo-A, Nogo-A-FL). Strikingly, TNFα appeared predominantly in trimers, i.e. under its most inflammatory form. f, g Additional antibodies directed against different parts of Nogo-A, named Rb1, S544, and 11C7, were used to determine the presence of Nogo-A proteins in the vitreous of NMDA-treated eyes. Increased levels of Nogo-A proteins were thus confirmed. Interestingly, proteins of similar molecular weight were found and may correspond to Nogo-A-F1, Nogo-A-F2, and Nogo-A-FL. Statistics: One-way ANOVA, Dunnett’s post hoc test, *P < 0.05; **P < 0.01; ***P < 0.001; †P < 0.0001. Scale bars: a, b close-up (bottom left) = 25 μm; b (top left) = 100 μm.
