Hemibrains were homogenized and divided into cytosolic and membrane fractions as previously described [72 (link),14 (link)]. For immunoblot analysis, 20 μg of total protein per lane was loaded on 4–12% Bis-Tris SDS-PAGE gels and blotted onto polyvinylidene fluoride (PVDF) membranes. To determine the effects of the immunotherapy in levels of α-syn, blotted samples from immunized α-syn tg mice were probed with antibodies against full length human α-syn (1:1000, SYN211, Life Technologies). Additional analysis was performed with antibodies against β-syn (Abcam). Incubation with primary antibodies was followed by species-appropriate incubation with secondary antibodies tagged with horseradish peroxidase (1:5000, Santa Cruz Biotechnology), visualization with enhanced chemiluminescence, and analysis with a Versadoc XL imaging apparatus (BioRad). Analysis of β-actin (Sigma) levels was used as a loading control.
To determine human α-syn levels, an ELISA analysis was used (Life Technologies), performed following manufacturer’s instructions. This ELISA kit is designed to react with human α-syn specifically, without detectable cross-reactivity to mouse α-syn.
To determine human α-syn levels, an ELISA analysis was used (Life Technologies), performed following manufacturer’s instructions. This ELISA kit is designed to react with human α-syn specifically, without detectable cross-reactivity to mouse α-syn.
