Anti ceruloplasmin

The following antibodies were used in this study: Anti-PrP antibodies 3F4 and 8H4 (Signet Laboratories, Dedham, MA), and SAF32 (189720, Cayman chemicals, USA), anti-ferritin (F5012, Sigma Aldrich, USA), anti-TfR (136800, Invitrogen, USA), anti-Tf (GTX2123, GeneTEX, USA), anti-ceruloplasmin (Dako, USA), Mouse IgG (5415, Cell signaling Technology, USA), anti-Fpn (NBP1-21502, Novus biological, USA), anti β-actin (MAB1501, Milipore, USA) and anti GAPDH (GT239, GeneTEX). HRP-conjugated anti-mouse, NA931V and anti-rabbit, NA934V (GE Healthcare, UK). Alexa Fluor-conjugated secondary antibodies (molecular probes), PNGase F (P0704S, NEB, USA). NAP (Non-animal protein) blocking solution (786-190T, G-Biosciences, USA), Hoechst (#33342, Invitrogen, USA), Fluoromount-G (Southern Biotech, USA).

For electrophoresis, equal amounts of serum (3 μl) were loaded per gel lane, size-fractionated on a 3–8% Tris-acetate-polyacrylamide gel (Invitrogen, Karlsruhe, Germany), and blotted onto a polyvinylidenediflouride (PVDF) membrane (Roche, Mannheim, Germany). For immunodetection, PVDF membranes were pretreated with I-Block buffer (Tropix, Bedford, MA, USA) followed by incubation with the following antibodies: mouse monoclonal anti-human Reelin (1:500; 142; Chemicon) or rabbit polyclonal anti-Ceruloplasmin (1:5000; Dako, Glostrup, Denmark) for 1 h at room temperature. After several washing steps, the membranes were incubated with the respective alkaline-phosphatase-conjugated secondary antibody (1:10.000; Tropix) for 1 h at room temperature and CDP Star (Tropix) was used as substrate for chemiluminescent detection by the Chemismart System (Peqlab Biotechnologies, Erlangen, Germany).
Quantification of Western blot signals was performed by optical density (OD) measurement using the Bio-1D software (Peqlab). OD values were determined for each of the three Reelin isoforms (400, 320 and 180 kDa) detected by the Reelin antibody. Data were normalized to Ceruloplasmin signals as loading control.