Fig 1: The prominent 35, 40, 45 kD autoantigens are identified as different GFAP isoforms. To test whether the 35, 40, 45 kD cluster seen in many plasma samples (A) is indeed caused by GFAP autoantibodies, the blot was reprobed with anti-GFAP antibody (B). Band Intensities from A and B were quantified and correlated (C) (p < 0.0001, linear regression). For abbreviations see Fig. 1
Fig 2: Identification of prominent autoantigens by 2D-Western Blot and MS-Sequencing. For the identification of the 45, 40, 35-kD cluster that is most prominent in the DM homogenate, DM homogenate was separated by 2D gel electrophoresis and blotted on a nitrocellulose membrane (B). The membrane was stained with a plasma sample that generated a strong 45, 40, 35 kD signal in the 1D Western Blot experiments (A). Proteins located at the strongest signals of the 2D-Western Blot were isolated and sequenced. The sequencing reaction identified the proteins underlying the spots A, B and C as GFAP (see also Table 2). C Reprobing of the membrane in B with anti-GFAP antibody. For abbreviations see Fig. 1
Fig 3: OGD-treated neuronal cell proliferation and apoptosis are modulated by E2F1/miR-122/SPRY2 axis. OGD-induced N2a cells were transfected with E2F1-OE, E2F1-OE + miR-122 Agomir, miR-122 Agomir, or miR-122 Agomir + SPRY2. (A) miR-122 expression after overexpression of E2F1 and miR-122, and SPRY2 expression after overexpression of miR-122 and SPRY2 determined in N2a cells by RT-qPCR. (B) N2a cell viability measured by GFAP staining. (C) N2a cell apoptosis assessed by flow cytometry. (D) Cell cycle distribution of N2a cells evaluated by flow cytometry. Unpaired t-test (Panel A) was conducted to compare two groups, whereas one-way (Panel B and C) or two-way ANOVA (Panel D) was carried out to compare multiple groups, followed by Tukey’s post hoc test. *p < 0.05 vs. N2a cells transfected with E2F1-OE, #p < 0.05 vs. N2a cells transfected with miR-122 Agomir.
Fig 4: Anti-GFAP autoantibodies preferentially recognize brain areas involved in early PD pathology. The three GFAP-bands (35, 40, 45 kD) from all 60 samples were quantified (A). Three blots were reprobed with anti-GFAP (B), showing the same pattern (p calculated with ANOVA and Friedmann test). Abbreviations: dorsal motor nucleus of the glossopharyngeal and vagal nerves (DM), substantia nigra (SN), anteromedial temporal mesocortex (MC), high-order sensory assoziation- and prefrontal fields (HC), first or sensory association- and premotor fields, primary sensoric and motoric fields (FC)
Fig 5: Comparison of anti-GFAP autoantibody titers of healthy controls and PD patients. Sum of the 35, 40, 45 KD GFAP bands for each brain area relative to the internal standard plasma (p calculated with Mann–Whitney U-test). Abbreviations: dorsal motor nucleus of the glossopharyngeal and vagal nerves (DM), substantia nigra (SN), anteromedial temporal mesocortex (MC), high-order sensory assoziation- and prefrontal fields (HC), first or sensory association- and premotor fields, primary sensoric and motoric fields (FC)
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