Journal: PLoS ONE

Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

doi: 10.1371/journal.pone.0064890

Figure Lengend Snippet: ( A ) Validation of the cdk1 sandwich ELISA assay performed with 10 fg of either GST (GST) or a chimeric protein containing the sequence of human cdk1 bound to GST (Cdk1-GST), and revealed with the anti-cdk1 PSTAIRE antibody. Normalized optical density (O.D.) at 405 nm is shown. ( B ) Validation of the phospho-cdk1 sandwich ELISA assay performed with cell lysates obtained from DCRNs cultured in the presence of 100 ng/ml NGF and 2 ng/ml BDNF, incubated either in the absence (- CIP) or presence (+ CIP) of CIP, and revealed with the anti-cdk1 (pTyr15) antibody. Normalized optical density (O.D.) at 405 nm is shown. ( C ) Cell lysates from DCRNs cultured in the presence (+) or absence (-) of the referred factors were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( D ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or K252a were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). *p<0.05; ***p<0.005 (Student’s t test; n = 3).

Article Snippet: K252a (200 nM) (Alomone Labs) and MK-1775 (300 nM) (Axon MedChem), both prepared in dimethyl sulfoxide, were added to the culture medium 10 min before BDNF treatment (DCRNs).

Techniques: Sandwich ELISA, Sequencing, Cell Culture, Incubation

Journal: Neural Plasticity

Article Title: Divergent Roles of p75 NTR and Trk Receptors in BDNF's Effects on Dendritic Spine Density and Morphology

doi: 10.1155/2012/578057

Figure Lengend Snippet: Role of Trk and p75 NTR in BDNF's effects on dendritic spine density and morphology. (a) Representative examples of dendritic segments of CA1 pyramidal neurons maintained in serum-containing media (SM) and treated with the function-blocking antibody of p75 NTR , REX (50 μ g/mL), and BDNF (250 ng/mL) for 48 hrs (scale bar represents 2 μ m). (b) Dendritic spine density expressed per 10 μ m of apical dendrite. (c) Proportion of each morphological type of dendritic spine, expressed as a fraction of the total spine population. (d) Representative examples of dendritic segments of CA1 pyramidal neurons maintained in SM and treated with k-252a (200 nM) and BDNF (250 ng/mL) for 48 hrs. (e) Dendritic spine density expressed per 10 μ m of apical dendrite. (f) Proportion of each morphological type of dendritic spines, expressed as a fraction of the total spine population. * P < 0.05, ** P < 0.01, and *** P < 0.001, after a one-way ANOVA.

Article Snippet: Slices were kept in a serum containing media throughout the course of the experiments and were randomly assigned to the following groups: (1) control, serum-containing media; (2) BDNF (250 ng/mL); (3) anti-p75 NTR antibody REX (50 μ g/mL), known to block p75 NTR function [ ] (provided by L. Reichardt, UCSF); (4) BDNF in the presence of REX antibody; (5) k-252a (200 nM, in DMSO; Calbiochem; San Diego, CA) to block autophosphorylation and activation of tyrosine kinase domains of plasma membrane neurotrophin receptors [ ]; (6) BDNF in the presence of k-252a; (7) TTX (1 μ M; Alomone Labs; Jerusalem, Israel); (8) TTX in the presence of k-252a.

Techniques: Blocking Assay

Journal: Neural Plasticity

Article Title: Divergent Roles of p75 NTR and Trk Receptors in BDNF's Effects on Dendritic Spine Density and Morphology

doi: 10.1155/2012/578057

Figure Lengend Snippet: Role of neuronal activity in k-252a's effects on dendritic spine density and morphology. (a) Representative examples of dendritic segments of CA1 pyramidal neurons maintained in serum-containing media (SM) and treated with k-252a (200 nM), TTX (1 μ M), or both k-252a and TTX for 48 hrs (scale bar represents 2 μ m). (b) Dendritic spine density expressed per 10 μ m of apical dendrite. (c) Proportion of each morphological type of dendritic spines, expressed as a fraction of the total spine population. * P < 0.05, ** P < 0.01, and *** P < 0.001, after a one-way ANOVA.

Article Snippet: Slices were kept in a serum containing media throughout the course of the experiments and were randomly assigned to the following groups: (1) control, serum-containing media; (2) BDNF (250 ng/mL); (3) anti-p75 NTR antibody REX (50 μ g/mL), known to block p75 NTR function [ ] (provided by L. Reichardt, UCSF); (4) BDNF in the presence of REX antibody; (5) k-252a (200 nM, in DMSO; Calbiochem; San Diego, CA) to block autophosphorylation and activation of tyrosine kinase domains of plasma membrane neurotrophin receptors [ ]; (6) BDNF in the presence of k-252a; (7) TTX (1 μ M; Alomone Labs; Jerusalem, Israel); (8) TTX in the presence of k-252a.

Techniques: Activity Assay

Journal: Neural Plasticity

Article Title: Divergent Roles of p75 NTR and Trk Receptors in BDNF's Effects on Dendritic Spine Density and Morphology

doi: 10.1155/2012/578057

Figure Lengend Snippet: Quantitative results of dendritic spine analyses .

Article Snippet: Slices were kept in a serum containing media throughout the course of the experiments and were randomly assigned to the following groups: (1) control, serum-containing media; (2) BDNF (250 ng/mL); (3) anti-p75 NTR antibody REX (50 μ g/mL), known to block p75 NTR function [ ] (provided by L. Reichardt, UCSF); (4) BDNF in the presence of REX antibody; (5) k-252a (200 nM, in DMSO; Calbiochem; San Diego, CA) to block autophosphorylation and activation of tyrosine kinase domains of plasma membrane neurotrophin receptors [ ]; (6) BDNF in the presence of k-252a; (7) TTX (1 μ M; Alomone Labs; Jerusalem, Israel); (8) TTX in the presence of k-252a.

Techniques:

Journal: Neural Plasticity

Article Title: Divergent Roles of p75 NTR and Trk Receptors in BDNF's Effects on Dendritic Spine Density and Morphology

doi: 10.1155/2012/578057

Figure Lengend Snippet: Summary of total dendritic length, cells, slices, and individual spines sampled for the quantitative dendritic spine analyses.

Article Snippet: Slices were kept in a serum containing media throughout the course of the experiments and were randomly assigned to the following groups: (1) control, serum-containing media; (2) BDNF (250 ng/mL); (3) anti-p75 NTR antibody REX (50 μ g/mL), known to block p75 NTR function [ ] (provided by L. Reichardt, UCSF); (4) BDNF in the presence of REX antibody; (5) k-252a (200 nM, in DMSO; Calbiochem; San Diego, CA) to block autophosphorylation and activation of tyrosine kinase domains of plasma membrane neurotrophin receptors [ ]; (6) BDNF in the presence of k-252a; (7) TTX (1 μ M; Alomone Labs; Jerusalem, Israel); (8) TTX in the presence of k-252a.

Techniques: