Journal: Molecular neurodegeneration

Article Title: Phosphorylation of collapsin response mediator protein-2 disrupts neuronal maturation in a model of adult neurogenesis: Implications for neurodegenerative disorders.

doi: 10.1186/1750-1326-6-67

Figure Lengend Snippet: Figure 2 Down-regulation of CDK5 reduces CRMP2 phosphorylation in NPC-derived neural progeny. Differentiating NPCs were treated on day 2 with the pharmacological inhibitor Roscovitine (Rosco) or siRNA against CDK5 (siCDK5), followed by infection with p35-adenovirus. On day 4, NPC-derived neural progeny were fixed for immunofluorescence or lysed for immunoblot analysis. (A-C) Immunocytochemical analysis with an antibody against pSer522-CRMP2 showed low levels of endogenous immunoreactivity in uninfected, vehicle-treated cells (A), with a slight reduction in cells treated with Roscovitine (B) or siCDK5 (C). (D-F) NPC-derived neural progeny expressing p35 showed a notable increase in CRMP2 phosphorylation (D) that was reversed by treatment with Rosco (E) or siCDK5 (F). (G) Reversal of CRMP2 hyperphosphorylation with CDK5 down-regulation. (H) Immunoblot analysis of pSer522-CRMP2, total (t)CRMP2, CDK5, and actin as a loading control. (I) Semi-quantitative image analysis of levels of CRMP2 phosphorylation in NPCs infected with p35-adv with or without Rosco or siCDK5. * p < 0.05 compared to vehicle-treated controls by one-way ANOVA with post-hoc Dunnett’s test. # p < 0.05 compared to p35-expressing NPCs by one-way ANOVA with post-hoc Tukey-Kramer test. Scale bar = 10 μm.

Article Snippet: For immunocytochemical analysis in human or mouse brain tissue, briefly as previously described [61] vibratome sections from the hippocampus (40 μm thick) of the HIV patients or from nontg, gp120 tg or CDK5+/mice were incubated with antibodies against phosphoCRMP2 (Ser522, 1:300, ECM Biosciences) or CRMP2 (1:300, Millipore).

Techniques: Phospho-proteomics, Derivative Assay, Infection, Immunofluorescence, Western Blot, Expressing, Control

Journal: Molecular neurodegeneration

Article Title: Phosphorylation of collapsin response mediator protein-2 disrupts neuronal maturation in a model of adult neurogenesis: Implications for neurodegenerative disorders.

doi: 10.1186/1750-1326-6-67

Figure Lengend Snippet: 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 b-tubulin immunofluorescence, or lysed for immunoblot analysis. (A-F) Immunocytochemical analysis of b-tubulin immunoreactivity showed reduced b-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 b-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).

Article Snippet: For immunocytochemical analysis in human or mouse brain tissue, briefly as previously described [61] vibratome sections from the hippocampus (40 μm thick) of the HIV patients or from nontg, gp120 tg or CDK5+/mice were incubated with antibodies against phosphoCRMP2 (Ser522, 1:300, ECM Biosciences) or CRMP2 (1:300, Millipore).

Techniques: Expressing, Mutagenesis, Construct, Derivative Assay, Transfection, Infection, Immunofluorescence, Western Blot, Plasmid Preparation, Phospho-proteomics