Figure 5.

NF186 expression is stabilized by interactions with ankyrin G. DRG neurons were nucleofected with either NF186-GFP or NF186ΔABD-GFP; half were maintained as neuron-only cultures (A), and the other half were seeded with Schwann cells after 1 wk and maintained in myelinating conditions (B). Detergent lysates were prepared at weekly intervals; fractionated by SDS-PAGE; blotted; probed for GFP, peripherin (as a loading control), and the myelin protein P0 (co-cultures); incubated with ¹²⁵I protein A; and analyzed via a phosphorimager. Quantitation is shown in Fig. S3 A (available at http://www.jcb.org/cgi/content/full/jcb.200612012/DC1). Full-length NF186-GFP is indicated (arrowhead); a proteolytic fragment of ∼150 kD (asterisk) is faintly visible in the neuron-only blots and prominently at initial co-culture time points. Addition of Schwann cells accelerates the turnover of NF186-GFP and, in particular, NF186ΔABD-GFP (compare corresponding time points in A and B). (C) DRG neurons expressing either NF186-GFP or NF186ΔABD-GFP under lentiviral control were fixed (control) or extracted with Triton X-100 and then fixed, followed by staining for GFP, P0, and ankyrin G. With extraction, NF186 staining is removed from nonmyelinated fibers but persists at most nodes in contrast to NF186ΔABD, which is removed from all nodes as well as nonmyelinated fibers. Insets show nodes and heminodes, indicated by arrowheads, at higher power. Bar, 20 μm.