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: Confocal sections from retinas of E6 chick embryos were double labeled with the anti-cdk1 specific mAbs (red) (B-6) ( A ), [A17] ( B ), and (17) ( C - E ), as well as with anti-p75 NTR (p75) ( C ), anti-phosphoHistone H3 (pH3) ( D ), or anti-TrkB ( E ) antibodies (green). Nuclear staining with bisbenzimide is shown in blue (Bisb.). ( A , B ) Cdk1 immunostaining is observed in the cytoplasm of a subpopulation of retinal cells and often in nuclei located apically (arrows). ( C ) A subset of cdk1-positive cells co-localize with p75 NTR (arrowhead), whereas many other p75 NTR -positive cells lack cdk1 immunolabeling (arrow). ( D ) Cdk1-positive cells co-localize with phosphoHistone H3 in the basal retina, close to the presumptive GCL (arrow). Arrowhead: apically located nucleus with cdk1-specific immunoreactivity. ( E ) TrkB-positive cells co-localize with cdk1 (arrow). Boxes: high magnification of the dashed areas. GCL: ganglion cell layer; PE: pigment epithelium; v: vitreous body. Bar: 20 µm ( A - D ), 40 µm ( E ).

Article Snippet: The mouse anti-cdk1 p34 (17) mAb (Santa Cruz Biotechnology) recognizes the sequence flanked by amino acids 224–230 of human origin (NNEVWPE; accession number: NP_001777) which is functionally conserved with the chick sequence.

Techniques: Labeling, Staining, Immunostaining, Immunolabeling

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 ) Lysates from DCRNs cultured for 20 h in the presence of different combinations of 100 ng/m NGF and 2 ng/ml BDNF were subjected to western blot with antibodies specific for either cdk1 (upper panel) or β-actin (lower panel). ( B ) Normalized cdk1/β-actin ratio. ( C ) Semiquantitative RT-PCR analysis of mRNA samples obtained from DCRNs cultured for 20 h in the presence of either vehicle (Control) or 2 ng/ml BDNF (BDNF). The levels of Cdk1 expression were normalized to Gapdh . N.S. non-significant; *p<0.05 (Student’s t test; n = 3–4).

Article Snippet: The mouse anti-cdk1 p34 (17) mAb (Santa Cruz Biotechnology) recognizes the sequence flanked by amino acids 224–230 of human origin (NNEVWPE; accession number: NP_001777) which is functionally conserved with the chick sequence.

Techniques: Cell Culture, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing

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 ) General cdk protein kinase activity, based on the phosphorylation of a Rb-derived peptide by the cdk activity present in cell extracts from DCRNs cultured for 20 h in the presence of 100 ng/ml NGF and treated for 30 min with either vehicle (Control) or 2 ng/ml BDNF. Equal amount of cell extracts were used in both cases. ( B ) Left panel, a representative western blot performed with an anti-cdk1 antibody using the cell extracts described above prior to immunoprecipitation (INPUT). Right panel, protein kinase activity immunoprecipitated with an anti cdk1 antibody (i.e. cdk1-specific kinase activity) from the cell extracts described above, normalized to the relative amount of cdk1 present in these extracts (see input in left panel). *p<0.05 (ANOVA; n = 3).

Article Snippet: The mouse anti-cdk1 p34 (17) mAb (Santa Cruz Biotechnology) recognizes the sequence flanked by amino acids 224–230 of human origin (NNEVWPE; accession number: NP_001777) which is functionally conserved with the chick sequence.

Techniques: Activity Assay, Derivative Assay, Cell Culture, Western Blot, Immunoprecipitation

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 ) E6 retinal cells electroporated with either EGFP (-) or cdk1 plus cyclin B1 (CC) and EGFP (+) were cultured under neurogenic conditions for 20 h in the presence of different combinations of 100 ng/ml NGF and 2 ng/ml BDNF. The percentage of mitotic figures was evaluated in the EGFP-positive cells. Lower panels show an example of a mitotic figure (pH3; red) in an EGFP-transfected cell (EGFP) (arrow). ( B ) E6 retinal cells were electroporated and cultured as above. The percentage of pyknotic nuclei was evaluated in the EGFP-positive cells. Lower panels show an example of a pyknotic nucleus in an EGFP-transfected cell (EGFP) (arrow). Bisb.: bisbenzimide. *p<0.05; ***p<0.005 (Student’s t test; n = 4). Bars: 10 µm.

Article Snippet: The mouse anti-cdk1 p34 (17) mAb (Santa Cruz Biotechnology) recognizes the sequence flanked by amino acids 224–230 of human origin (NNEVWPE; accession number: NP_001777) which is functionally conserved with the chick sequence.

Techniques: Cell Culture, Transfection

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: The mouse anti-cdk1 p34 (17) mAb (Santa Cruz Biotechnology) recognizes the sequence flanked by amino acids 224–230 of human origin (NNEVWPE; accession number: NP_001777) which is functionally conserved with the chick sequence.

Techniques: Sandwich ELISA, Sequencing, Cell Culture, Incubation

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 ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or 300 nM MK-1775 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). ( B ) Cell lysates from CEFs cultured in the presence (+) or absence (-) of 300 nM MK-1775 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<0.005 (Student’s t test; n = 3). # p<0.05 (NGF/BDNF/MK-1775 vs NGF/MK-1775; Student’s t test; n = 3).

Article Snippet: The mouse anti-cdk1 p34 (17) mAb (Santa Cruz Biotechnology) recognizes the sequence flanked by amino acids 224–230 of human origin (NNEVWPE; accession number: NP_001777) which is functionally conserved with the chick sequence.

Techniques: Cell Culture, Sandwich ELISA

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: E6 retinal cells electroporated with either EGFP (-) or the Tyr15Phe mutant form of cdk1 plus cyclin B1 (Mut CC) and EGFP (+) were cultured for 20 h under neurogenic conditions in the presence of different combinations of 100 ng/ml NGF and 2 ng/m BDNF. The percentage of pyknotic nuclei was evaluated in EGFP-positive cells. *p<0.05 (Student’s t test; n = 4).

Article Snippet: The mouse anti-cdk1 p34 (17) mAb (Santa Cruz Biotechnology) recognizes the sequence flanked by amino acids 224–230 of human origin (NNEVWPE; accession number: NP_001777) which is functionally conserved with the chick sequence.

Techniques: Mutagenesis, Cell Culture

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: TrkB activation by BDNF prevents the increase cyclin B and cdk1 protein expression (dashed line) and induces phosphorylation of cdk1 at Tyr15 (thick black arrow), thus inhibiting cdk1 kinase activity. This effect participates in the G2/M arrest (grey line) observed in tetraploid RGCs.

Article Snippet: The mouse anti-cdk1 p34 (17) mAb (Santa Cruz Biotechnology) recognizes the sequence flanked by amino acids 224–230 of human origin (NNEVWPE; accession number: NP_001777) which is functionally conserved with the chick sequence.

Techniques: Activation Assay, Expressing, Activity Assay

Journal: Oncogene

Article Title: Human and mouse cyclin D2 splice variants: transforming activity and subcellular localization.

doi: 10.1038/sj.onc.1210750

Figure Lengend Snippet: Figure 4 Analysis of the Myc-tagged truncated cyclin D2 binding with cyclin-dependent kinase 4 (CDK4) and phosphorylation of pRb. (a) For co-immunoprecipitations, NIH/3T3 mock transfected (NIH/3T3), transfected with the pCMV-Myc vector, the Myc- truncated cyclin D2 construct (Myc-D2Trc) and the Myc-cyclin D2 construct (Myc-D2) were used. Immunoprecipitation (IP) was performed with anti-CDK4 antibodies and 1 mg of protein from the whole-cell lysate. The upper panel shows the precipitated Myc- tagged truncated cyclin D2 and the Myc-tagged cyclin D2 detected by western blot analysis with a Myc epitope antibody. The lower panel shows the endogenous expression levels of CDK4 in the different transfected cells used for IP. (b) For kinase assays, NIH/3T3 cells were transfected with pCMV-Myc vector, Myc-cyclin D2 and Myc-truncated cyclin D2. Lysates were prepared and subjected to immune precipitation using anti-Myc antibody. Precipitates were tested for in vitro kinase activity using glutathione S-transferase (GST)-Rb C0-terminus as substrate. Kinase reactions were analysed by SDS–PAGE followed by exposure of the gel on X-ray film (top panel). The gel was stained with Coomassie Brilliant Blue to visualize the substrate (GST-Rb) in each lane. Whole-cell lysates corresponding to each lane were analysed by immunoblot with anti-CDK4 and c-Myc antibody. (c) In vitro CAK phosphorylation of cyclin D2–CDK4 complexes. NIH/3T3 cell lysates containing CDK4 assembled with either Myc-cyclin D2 or Myc-D2Trc were precipitated with anti-Myc antibodies. Suspended immune complexes were used as substrates for recombinant CAK (CDK7/cyclin H/MAT1 produced in Sf21 cells), and 32P-labeled complexes were denaturated and separated on polyacrylamide gels. The position of phosphorylated CDK4 is indicated on the left. Autoradiographic exposure time was 3 h (top panel). A portion of each cell lysate used in the CAK assay was precipitated with anti-Myc antibodies, separated on gel, blotted to PVDF membrane, probed with anti-CDK4 antibodies and visualized by enhanced chemiluminescence.

Article Snippet: The supernatants were precipitated for 4 h at 4 1C with protein A-Sepharose beads (GE Healthcare) alone or precoated with saturating amounts of the anti-CDK4 antibody (Santa Cruz sc-601, Santa Cruz, CA, USA).

Techniques: Binding Assay, Phospho-proteomics, Transfection, Plasmid Preparation, Construct, Immunoprecipitation, Western Blot, Expressing, In Vitro, Activity Assay, SDS Page, Staining, Recombinant, Produced, Labeling, Membrane