Horseradish peroxidase

For each biological replicate, 10 μg of protein were solubilized in 4x SDS-polyacrylamide gel electrophoresis sample buffer and separated on SDS gels containing 10% acrylamide. Subsequently, they were blotted on nitrocellulose membrane (HP42.1, Roth). To block non-specific binding sites after blotting, the membrane was incubated with 5% dried milk in TBS-Tween 20 for 1 h. After blocking, the membranes were incubated overnight at 4°C with the primary monoclonal antibody α5 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA). Since only membrane proteins were isolated from transfected cells, detection of the α subunit also indicates the presence of the β subunit. The primary antibody was detected using a goat-anti-mouse secondary antibody conjugated with horseradish peroxidase (Dianova, Hamburg, Germany). The staining of the precipitated polypeptide-antibody complexes was performed by addition of 60 mg 4-chloro-1 naphtol (Sigma-Aldrich, Taufkirchen, Germany) in 20 ml ice-cold methanol to 100 ml phosphate buffered saline (PBS) containing 60 μl 30% H₂O₂. See S8 Fig.

Cells were washed with ice cold PBS and scraped off in lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing protease and phosphatase inhibitors (Complete Mini EDTAfree and PhosStop, both Roche Applied Science). Sciatic nerves and whole brains of mice postnatal day 18 (P18) were dissociated in lysis buffer using a TissueRuptor (Qiagen). The lysates were incubated on a rotating wheel at 4°C for 45 min and afterwards cleared from nuclei and debris by centrifugation at 2000x g and 4°C for 5 min.
Separation of proteins were performed by SDS-PAGE using a Mini PROTEAN system (Bio-Rad) or Novex NuPAGE SDS-PAGE Gel system (Life technologies) and transferred onto Roti-PVDF membranes (0.45μm, Roth) using a Mini TransBlot Electrophoretic Transfer Cell device (Bio-Rad). Precision Plus Dual Color Protein Standard (Bio-Rad) was used as a marker. Membranes were blocked with 4% (w/v) milk (Roth) in TBST (50 mM Tris, 150 mM NaCl, pH 7.2, 0.1% (v/v) Tween 20) for 30 min at room temperature. Binding of primary antibodies was carried out either at 4°C over night or for 1h at room temperature. Suitable secondary antibodies (coupled to horseradish peroxidase, Dianova) were incubated for 30min at room temperature. All antibodies were diluted in blocking medium. Bands were analyzed densitometrically using ImageLab Software (Biorad) and MBP Signals were normalized to CNP Signals.

Cells were harvested by scraping in cold lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA pH 7.4, 1% (v/v) Nonidet P-40, 0.25% (w/v) sodium deoxycholate) containing HALT protease and phosphatase inhibitors (Thermo Scientific) and incubating on a rotating wheel at 4°C for 45 min. The lysate was cleared from nuclei and debris by centrifugation at 300×g and 4°C for 10 min.
Proteins were separated by SDS-PAGE using a Mini PROTEAN 3 system (Bio-Rad) and transferred onto PVDF membranes (Immobilion-P, Millipore) using a Mini TransBlot Electrophoretic Transfer Cell device (Bio-Rad). To block unspecific binding, membranes were incubated with 4% (w/v) milk (Roth) in TBST (0.05 M Tris, 0.15 M NaCl, pH 7.2, 0.1% (v/v) Tween20) for 30 min at room temperature. Primary antibody binding was carried out overnight at 4°C and appropriate secondary antibodies (coupled with horseradish peroxidase, Dianova) were incubated for 30 min at room temperature, both in blocking medium.