Myelin preparation and GST-Nogo-66 expression.
(A) Immunofluorescence analysis of Nestin (green) in the primary culture NPCs. Nuclei were stained by PI (shown in red). Nearly all of the cells were immunostaining positive for Nestin. (B) Western blot analysis detecting Nogo-A and MBP proteins in myelin preparation with a Nogo-A antibody and a MBP antibody. In myelin preparation from adult rat spinal cord not only MBP, a major myelin component, was detected, but Nogo-A was also detected. (C) GST-Nogo-66 SDS-PAGE image. Soluble GST-Nogo-66 purified by glutathione-resin was broken and contained about 30% full-length GST-Nogo-66. Full-length GST-Nogo-66 was about 95% in the inclusion bodies. (D) In vitro analysis of renatured GST-Nogo-66 biological acitivity. Postnatal day 8 rat cerebellar granule neurons were dissociated and placed in culture on slides coated with poly-L-lysine and later supplemented with GST or GST-Nogo-66. After culturing for 48 hours, cells were fixed, permeabalized and stained with a β III tubulin antibody (green) and Nuclei were stained by Hoechst 33342 (blue). Micrographs of the treated cultures showed the inhibitory effects of GST-Nogo-66. (E) Dose-response curve of CGCs treated as in (D) of GST-Nogo-66 protein against neurite length. Results represent the mean neurite length per cell from two independent experiments, with more than 50 cells being measured in three separate wells for each treatment. GST-Nogo-66 significantly inhibited neurite outgrowth at 50 nM concentration (2-tailed t test, P<0.01).