Anti 3 nt

Purified protein stocks (ASYN variants and GFP) were stored in H₂O, because of a lower tendency to form covalent ASYN di- and multimers compared with storage in buffers. Samples were directly subjected to separation by a 15% SDS gel (2 μg/lane), transferred onto nitrocellulose membranes (Amersham Biosciences) and blocked with 5% milk powder in PBS-Tween (0.1%) for 1 h. Monoclonal antibodies (anti-ASYN, 1:3000, BD Bioscience; anti-3-NT, 1:250, Hycult Biotech; anti-3-NT Sigma, 1:250; anti-C-terminal amino acid sequence EPEA antibody fragment (CaptureSelect™ Biotin Anti-C-tag Conjugate, 1:1000, Thermo Fisher) were incubated over night at 4 °C. The horseradish peroxidase-conjugated secondary antibody (goat anti mouse IgG, Jackson ImmunoResearch, 1:2500), or Streptavidin-HRP (1:5000; Invitrogen) was incubated for 1 h. Protein bands were detected by a FUSION SL™ system (Peqlab, Erlangen, Germany) and quantified by ImaEva.