Fig. 7. Chase of surface biotinylated PLP, MAG and MOG through endocytosis and recycling. Primary cultured oligodendrocytes were cell-surface biotinylated at 4°C and incubated for 0, 4 and 24 hours at 37°C. (A) Streptavidin-FITC staining of permeabilized cells to visualize cell-surface localization (0 hours, arrow), endocytosis (4 hours, arrowhead), and recycling of biotinylated proteins (24 hours, asterisks; see also supplementary material Fig. S4). Within 24 hours, biotinylated proteins recycle from the cell body to the periphery where they localize to myelin-like membrane sheets (asterisks). 3D stacks of confocal planes are shown. Scale bars: 5 µm. (B) Subfractionation of primary oligodendrocytes as described in Fig. 6 directly after biotinylation (0 hours) and after a chase period of endocytosis and recycling for 24 hours at 37°C. Biotinylated proteins were precipitated from the fractions using NeutrAvidin-beads and analyzed by western blotting (a). Within 24 hours, biotinylated PLP and MOG significantly shift from heavy (H) to light fractions (L). MAG remains associated with the heavy (H) fraction. As expected, endosomal syntaxin 6 is not biotinylated and does not precipitate with NeutrAvidin (negative control). Western blots of the total fractions are also shown (b). The distribution of total PLP, MAG and MOG between the fractions does not significantly change over 24 hours at 37°C. Note that gels were run under nonreducing conditions, favoring dimeric PLP. (C) Densitometric quantification of NeutrAvidin-precipitated proteins from three independent experiments. The relative distribution of biotinylated PLP (monomeric + dimeric), MAG and MOG between the fractions is shown as a percentage. Error bars represent s.e.m.; ^*P<0.05; ^**P<0.01 (paired t-test).