Western blot analysis

Myelin proteins were analysed in samples of purified myelin (see above) or in whole brain extracts. Whole mouse brain was homogenized at 10% (w/v) in 10 nM Tris–HCl, pH 7.4, 0.32 M sucrose and a protease inhibitor coctail (Sigma). Protein concentration was determined by BCA protein assay kit (Ornat). Protein samples (15 μg) were loaded on either a 13.5% or a 10% SDS-PAGE, and following electrophoresis, proteins were transferred to a PVDF membrane (Biorad, Petach Tikva, Israel). The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline pH 8.0 (10 mM Tris–HCl, 150 mM NaCl, pH 8.0) containing 0.1% Tween-20 (TBST) for 1 h. The membrane was then incubated at 4 ⁰C for 16–18 h in 1% non-fat dry milk in TBST and a specific antibody. The following antibodies were used at the specified concentrations: anti-rat MBP mAb (1:1500, Serotec, Hercules, CA, USA); anti-rabbit MAG mAb (1:1000, Cell Signaling Technology, Danvers, MA, USA); anti-mouse CNPase mAb (1:500, Sigma); anti-mouse MOG mAb (1:5000, Merck-Millipore, Darmstadt, Germany); anti-rabbit PLP/DM20 pAb (1:1000, Novus Biologicals, Littleton, CO, USA); anti-rat tubulin mAb (1:10,000, AbDSerotec); and anti-P25α pAb (1:500, a gift from Paul Henning Jensen, Aarhus University, Denmark). Immunoreactive bands were detected with HRP-conjugated donkey anti-mouse (1:10,000), goat anti-rat (1:10,000), or goat anti-rabbit (1:10,000) secondary antibody. The signal was visualized with EZ-ECL (Biological Industries, Beit Haemek, Israel), scanned with a Umax Magic Scan (Eastman Kodak, Rochester, NY, USA) and analyzed for density of each signal using UN-SCAN-IT GEL 3.1 software (Silk Scientific, Orem, UT, USA). The signal obtained for each protein in a specific sample of myelin was normalized to the total amount of tubulin protein in the same sample.