Impaired neurite outgrowth is accompanied by CRMP2 hyperphosphorylation in NPC-derived neural progeny overexpressing p35. Differentiating NPCs were grown on glass coverslips and infected on day 2 with an adenoviral vector expressing p35. Cells were processed for immunocytochemistry and immunoblot analyses. For β-tubulin immunofluorescence, on day 4, cells were briefly extracted, then fixed with glutaraldehyde and processed. (A,B) Reduced β-tubulin-immunoreactive neurites in NPC-derived neural progeny infected with p35-adv. (C) Quantitative image analysis showing reduced neurite lengths in NPC-derived neural progeny with p35 expression. (D) Immunoblot analysis of cell lysates from control and p35-expressing NPCs using a panel of antibodies specific for various isoforms and phosphorylated forms of CRMP. (E) Immunoblot analysis of total cell lysates showing increased CRMP2 phosphorylation in NPC-derived neural progeny expressing p35 from adenoviral (left panel) or plasmid (right panel) vectors. (F) Semi-quantitative image analysis of fold change in CRMP2 phosphorylation at the CDK5 epitope (Ser522) in immunoblots from cells expressing p35 from adenoviral or plasmid vectors. * p < 0.05 compared to vehicle-treated controls by one-way ANOVA with post-hoc Dunnett's test. Scale bar = 10 μm.