Figure 3

Expression of S522A-CRMP2 mutant construct rescues neurite deficits in p35-expressing NPC-derived neural progeny. Differentiating NPCs were transfected on day 2 with wild-type (WT) human CRMP2 or mutated S522A-CRMP2 plasmids, followed by infection with p35 adenovirus. Cells were fixed on day 4 of differentiation with glutaraldehyde for β-tubulin immunofluorescence, or lysed for immunoblot analysis. (A-F) Immunocytochemical analysis of β-tubulin immunoreactivity showed reduced β-tubulin-positive neurites in NPC-derived neural progeny infected with p35-adv with or without co-expression of WT-CRMP2, and rescue with co-expression of S522A-CRMP2 plasmid. (G) Image analysis showing reduced β-tubulin-positive neurite lengths in p35-expressing and p35+WT-CRMP2 NPC-derived neural progeny that was recovered after co-expression of p35 and S522A-CRMP2. (H) Immunoblot analysis showing levels of CRMP2 phosphorylation in p35-expressing NPC-derived neural progeny transfected with S522A-CRMP2 or WT-CRMP2 plasmid. (I) Semi-quantitative image analysis of ratios of pSer522-CRMP2 (pCRMP2)/total CRMP2 (tCRMP2) levels in NPC-derived neural progeny expressing p35 with and without WT-CRMP2 or S522A-CRMP2 co-expression. * p < 0.05 compared to vehicle-treated controls by one-way ANOVA with post-hoc Dunnett's test. # p < 0.05 compared to WT-CRMP2-transfected NPCs by one-way ANOVA with post-hoc Tukey-Kramer test. Scale bar = 25 μm, 10 μm (insets).