Assays

Mice were sacrificed by cervical dislocation (n=4) and brains were subsequently extracted and the injury-site and corresponding area on the contralateral side was removed using a 2.5 mm biopsy punch and the white matter was removed. Samples were homogenized using a (100 μl) dounce (Wheaton #357844) in 100 ul PBS (with protease inhibitor cocktail and Ethylenediaminetetraacetic acid (EDTA)) and directly frozen in liquid nitrogen and stored in -80 °C until tissue protein fractionation. Following ...

Cells were collected and lysed in urea buffer (9 M Urea, 50 mM Tris pH 8, 150 mM NaCl, 1x Roche protease inhibitor cocktail) followed by short sonification. Samples were cleared by centrifugation and protein amounts were adapted. Protein reduction was performed with dithiothreitol (DTT; 5 mM final) for 25 min at 56°C and protein alkylation by the addition of iodoacetamide (14 mM final) for 30 min at room temperature. Protein mixtures were quenched with DTT and diluted 1:5 with 1 M Tris-Hcl, pH ...

Immunoprecipitation: Cells grown in 2-4x15 cm cell culture plates per sample were harvested by scraping on ice and stored at -80. Lysis was performed for 30 min at 4°C with MCLB buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 0.5% NP40, 1x PhosStop,1x protease inhibitor) or Glycerol buffer (20 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 0.5% TritonX, 1x PhosStop, inhibitor, 1x protease inhibitor). Samples were cleared from debris by centrifugation (20.000 g for 10 min at 4°C) and Ultrafree®-CL ...

Pellets of membrane-protected material were lysed with RIPA buffer containing quenchers (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, 1x cOmplete Protease Inhibitor Cocktail (Roche), 1x PhosSTOP 956 (Roche), 10 mM sodium ascorbate, 1 mM Trolox and 1 mM sodium azide), sonicated and centrifuged at 10,000x g for 10 min. The supernatant was incubated with Streptavidin-agarose (Sigma-Aldrich) overnight, which was balanced with RIPA buffer containing quenchers. After ...