Fig. 7.

Netrin-1 is enriched in periaxonal myelin. A, The flow chart illustrates the origin of the fractions containing purified myelin, periaxonal myelin, and axolemma. Each step is labeled with the number corresponding to the lane on the gel in B containing that fraction. B, The proteins MBP (∼14 kDa), CNP (∼46 kDa), MAG (∼100 kDa), and NFM (∼145 kDa) were used as markers for the enrichment of purified myelin (compact myelin), periaxonal myelin, and axolemmal membranes, respectively. Full-length netrin-1 protein partitioned between the crude myelin (lane 2) and crude gray matter (lane 3) fractions. After separation of the crude white matter fraction into myelin (lane 4) and crude periaxolemma (lane 5), netrin-1 (∼75 kDa) was enriched in the crude periaxolemmal fraction. No netrin immunoreactivity was detected in the purified myelin fraction containing compact myelin (lane 6). After further fractionation of the crude periaxolemmal myelin, full-length netrin-1 partitioned between the fractions enriched for periaxonal myelin (lane 7) and axolemma (lane 8). A 10% and a 12% acrylamide gel were used to separate ∼20 μg of total protein loaded per lane to visualize the different markers shown. Below the immunoblots, a 12% gel containing these fractions is shown stained with Coomassie blue, illustrating the distribution of proteins in each fraction and that comparable amounts of protein are present in each lane. Molecular weight markers correspond to 116, 97.4, 66.2, 45, 31, and 21 kDa.