Journal: The Journal of Neuroscience

Article Title: Decreased Cyclin-Dependent Kinase 5 (cdk5) Activity Is Accompanied by Redistribution of cdk5 and Cytoskeletal Proteins and Increased Cytoskeletal Protein Phosphorylation in p35 Null Mice

doi: 10.1523/JNEUROSCI.23-33-10633.2003

Figure Lengend Snippet: Antibodies

Article Snippet: Protran nitrocellulose membrane for immunoblotting was purchased from Intermountain Scientific (Kaysville, UT). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Group Antibody Isotype Epitope Western dilution IHC dilution Source cdk5 cdk5 (C-8) Rabbit polyclonal cdk5 C terminus 1:100 1:100 Santa Cruz Biotechnology p35 p35 (C-19) Rabbit polyclonal p35 C terminus 1:100 nd Santa Cruz Biotechnology p67 p67 Rabbit polyclonal p67 N terminus 1:5000 nd H. Pant AP18 Mouse IgG1 MAP2B phosphorserine 136 1:1000 1:1000 L. Binder MAP2 AP20 Mouse IgG1 Total MAP2A, 2B and 2C 1:1000 1:500 Sigma R39 Rabbit polyclonal Total NF-H, NF-M and NF-L 1:2000 nd H. Pant SMI31 Mouse IgG1 Phospho-NF-H and NF-M 1:1000 1:500 Sternberger Monoclonals SMI34 Mouse IgG1 Phospho-NF-H 1:1000 nd Sternberger Monoclonals NFs SMI32 Mouse IgG1 Nonphosphorylated NF-H 1:500 1:500 Sternberger Monoclonals TG5 Mouse IgG1 Total tau 1:10 nd I. Vincent CP22 Mouse IgM Tau phosphothreonine 175 1:10 nd P. Davies CP13 Mouse IgG1 Tau phosphoserine 202 1:10 1:10 P. Davies MC6 Mouse IgG1 Tau phosphoserine 235 1:10 nd P. Davies MC15 Mouse IgM Tau phosphoserine 235 1:10 nd P. Davies Tau PHF1 Mouse IgG1 Tau phosphoserine 396/404 1:20 1:10 P. Davies Neuronal antigen NeuN Mouse IgG1 Neuronal nuclei 1:250 nd Chemicon Syntaxin SP6 Mouse IgG1 Syntaxin 1:20 1:10 W. Honer GSK3β 9332 Rabbit polyclonal Total GSK3β 1:500 nd Cell Signaling Technology β-Catenin 9562 Rabbit polyclonal Total β-catenin 1:500 nd Cell Signaling Technology Open in a separate window nd, Not determined.

Techniques: Western Blot

Journal: The Journal of Neuroscience

Article Title: Decreased Cyclin-Dependent Kinase 5 (cdk5) Activity Is Accompanied by Redistribution of cdk5 and Cytoskeletal Proteins and Increased Cytoskeletal Protein Phosphorylation in p35 Null Mice

doi: 10.1523/JNEUROSCI.23-33-10633.2003

Figure Lengend Snippet: Distribution of MAP2 in p35+/+ and p35-/- brains. Images of 10 μm sagittal sections of p35+/+ (A, C, E, G, I, K) and p35-/- (B, D, F, H, J, L) brains incubated with the AP18 antibody to phospho-Ser136 in MAP2B (A, B, E, F, I, J) or the AP20 nonphospho-MAP2 antibody (C, D, G, H, K, L). A, E, I, Phospho-MAP2B (AP18) stains neuronal somata (arrows) and apical dendrites (arrowheads) in p35+/+ cortex (A), hippocampus (E), and pons (I). B, F, J, In p35-/- brains, AP18 staining is diminished greatly or not detected in neuronal somata (arrows) and dendrites (arrowheads) in the cortex (B), hippocampus (F), and pons (J). Dendritic organization is disrupted (B, F), and staining is sometimes shifted to the neuropil (F, asterisk). The granule cell layer of the CA2/CA3 region of the hippocampus is disorganized in p35-/- brains (F). C, G, K, The AP20 nonphospho-MAP2 antibody stains neuronal somata (arrows) and apical dendrites (arrowheads) in p35+/+ cortex (C), hippocampus (G), and cerebellum (K). D, H, L, In p35-/- brains, AP20 staining is shifted from neuronal somata and apical dendrites to more distal processes (asterisks), and dendritic organization is disrupted in cortex (D) and hippocampus (H). Disorganization of the granule cell layer is apparent (H). In the cerebellum (L), AP20 staining in Purkinje cell somata is decreased (arrow), whereas staining in more distal dendritic arborizations is robust.

Article Snippet: Protran nitrocellulose membrane for immunoblotting was purchased from Intermountain Scientific (Kaysville, UT). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Group Antibody Isotype Epitope Western dilution IHC dilution Source cdk5 cdk5 (C-8) Rabbit polyclonal cdk5 C terminus 1:100 1:100 Santa Cruz Biotechnology p35 p35 (C-19) Rabbit polyclonal p35 C terminus 1:100 nd Santa Cruz Biotechnology p67 p67 Rabbit polyclonal p67 N terminus 1:5000 nd H. Pant AP18 Mouse IgG1 MAP2B phosphorserine 136 1:1000 1:1000 L. Binder MAP2 AP20 Mouse IgG1 Total MAP2A, 2B and 2C 1:1000 1:500 Sigma R39 Rabbit polyclonal Total NF-H, NF-M and NF-L 1:2000 nd H. Pant SMI31 Mouse IgG1 Phospho-NF-H and NF-M 1:1000 1:500 Sternberger Monoclonals SMI34 Mouse IgG1 Phospho-NF-H 1:1000 nd Sternberger Monoclonals NFs SMI32 Mouse IgG1 Nonphosphorylated NF-H 1:500 1:500 Sternberger Monoclonals TG5 Mouse IgG1 Total tau 1:10 nd I. Vincent CP22 Mouse IgM Tau phosphothreonine 175 1:10 nd P. Davies CP13 Mouse IgG1 Tau phosphoserine 202 1:10 1:10 P. Davies MC6 Mouse IgG1 Tau phosphoserine 235 1:10 nd P. Davies MC15 Mouse IgM Tau phosphoserine 235 1:10 nd P. Davies Tau PHF1 Mouse IgG1 Tau phosphoserine 396/404 1:20 1:10 P. Davies Neuronal antigen NeuN Mouse IgG1 Neuronal nuclei 1:250 nd Chemicon Syntaxin SP6 Mouse IgG1 Syntaxin 1:20 1:10 W. Honer GSK3β 9332 Rabbit polyclonal Total GSK3β 1:500 nd Cell Signaling Technology β-Catenin 9562 Rabbit polyclonal Total β-catenin 1:500 nd Cell Signaling Technology Open in a separate window nd, Not determined.

Techniques: Incubation, Staining

Journal: The Journal of Neuroscience

Article Title: Decreased Cyclin-Dependent Kinase 5 (cdk5) Activity Is Accompanied by Redistribution of cdk5 and Cytoskeletal Proteins and Increased Cytoskeletal Protein Phosphorylation in p35 Null Mice

doi: 10.1523/JNEUROSCI.23-33-10633.2003

Figure Lengend Snippet: Western blots demonstrating phosphorylation status of cytoskeletal proteins in p35+/+ and p35-/- mouse brains. The amount of total protein in supernatants from whole-brain homogenates is shown in parentheses for each antibody used. Blots incubated with an antibody to neuronal antigens, NeuN, are shown as loading controls. A, NFs. Total levels of NF-H and NF-M are similar in p35+/+ and p35-/- brains (R39; 10 μg). Phosphorylation of NF-H at sites in the C terminus but outside the Lys-Ser-Pro repeat region is lower in p35-/- brains, as shown by an increase in bands detected by an antibody to only nonphosphorylated NF-H (SMI32; 50 μg). Phosphorylation of NF-H at repetitive Lys-Ser-Pro motifs is similar in p35+/+ and p35-/- brains, but phosphorylation of these repetitive motifs is increased in NF-M in p35-/- brains (SMI31; 5 μg). Phosphorylation of NF-H at other sites in the C-terminal repeat region is increased in p35-/- brains (SMI34; 5 μg). B, MAP2. Similar levels of total MAP2A and 2B are detected in p35+/+ and p35-/- brains (AP20; 10 μg), but there is a significant increase in phospho-MAP2B levels in p35-/- brains (AP18; 25 μg). C, Tau. Total tau levels are increased in p35-/- brains compared with p35+/+ brains (TG5; 25 μg). Phosphorylation of tau increases at Ser202 (CP13; 25 μg), Ser235 (MC15; 25 μg), and Ser 396/404 (PHF-1; 25 μg) in p35-/- brains. D, Antibodies to phosphotau epitopes recognize an epitope in a protein that migrates at the same rate as NF-H, and levels of these phosphoepitopes increase in p35-/- brains.

Article Snippet: Protran nitrocellulose membrane for immunoblotting was purchased from Intermountain Scientific (Kaysville, UT). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Group Antibody Isotype Epitope Western dilution IHC dilution Source cdk5 cdk5 (C-8) Rabbit polyclonal cdk5 C terminus 1:100 1:100 Santa Cruz Biotechnology p35 p35 (C-19) Rabbit polyclonal p35 C terminus 1:100 nd Santa Cruz Biotechnology p67 p67 Rabbit polyclonal p67 N terminus 1:5000 nd H. Pant AP18 Mouse IgG1 MAP2B phosphorserine 136 1:1000 1:1000 L. Binder MAP2 AP20 Mouse IgG1 Total MAP2A, 2B and 2C 1:1000 1:500 Sigma R39 Rabbit polyclonal Total NF-H, NF-M and NF-L 1:2000 nd H. Pant SMI31 Mouse IgG1 Phospho-NF-H and NF-M 1:1000 1:500 Sternberger Monoclonals SMI34 Mouse IgG1 Phospho-NF-H 1:1000 nd Sternberger Monoclonals NFs SMI32 Mouse IgG1 Nonphosphorylated NF-H 1:500 1:500 Sternberger Monoclonals TG5 Mouse IgG1 Total tau 1:10 nd I. Vincent CP22 Mouse IgM Tau phosphothreonine 175 1:10 nd P. Davies CP13 Mouse IgG1 Tau phosphoserine 202 1:10 1:10 P. Davies MC6 Mouse IgG1 Tau phosphoserine 235 1:10 nd P. Davies MC15 Mouse IgM Tau phosphoserine 235 1:10 nd P. Davies Tau PHF1 Mouse IgG1 Tau phosphoserine 396/404 1:20 1:10 P. Davies Neuronal antigen NeuN Mouse IgG1 Neuronal nuclei 1:250 nd Chemicon Syntaxin SP6 Mouse IgG1 Syntaxin 1:20 1:10 W. Honer GSK3β 9332 Rabbit polyclonal Total GSK3β 1:500 nd Cell Signaling Technology β-Catenin 9562 Rabbit polyclonal Total β-catenin 1:500 nd Cell Signaling Technology Open in a separate window nd, Not determined.

Techniques: Western Blot, Phospho-proteomics, Incubation

Journal: The Journal of Neuroscience

Article Title: Decreased Cyclin-Dependent Kinase 5 (cdk5) Activity Is Accompanied by Redistribution of cdk5 and Cytoskeletal Proteins and Increased Cytoskeletal Protein Phosphorylation in p35 Null Mice

doi: 10.1523/JNEUROSCI.23-33-10633.2003

Figure Lengend Snippet: Distribution of cytoskeletal protein phosphoepitopes relative to cdk5. Images of 10 μm sagittal sections from p35-/- (A-C) and p35+/+ (D-I) brains incubated with antibodies to cdk5 and antibodies to cytoskeletal protein phosphoepitopes. A-C, In p35-/- cortex, cdk5 (green) colocalizes with non-P-NF-H (A, SMI32, red), P-NF-H/M (B, SMI31, red), and phosphotau (C, PHF1, red) in the abnormal fascicles (arrows) characteristic of these mice. D, E, In p35+/+ cortex, axons display immunoreactivity for cdk5 (D, E, green arrows), phospho-NF-H/M (D, SMI31, red arrowheads), and phosphotau (E, PHF-1, red arrowheads), but there is little, if any, overlap in the distribution of cdk5 with either phosphoepitope. F-H, In p35+/+ mice, large myelinated fiber tracts in the basal ganglia (arrows) are negative for cdk5 staining (F) but heavily stained for P-NF-H/M (G-SMI31) and phosphotau (H-PHF-1). I, In p35+/+ cortex, cdk5 (green) and phospho-MAP2B (AP18, red) both stain neuronal somata, but AP18 segregates to apical dendrites (arrow) and cdk5 to proximal axons (arrowhead).

Article Snippet: Protran nitrocellulose membrane for immunoblotting was purchased from Intermountain Scientific (Kaysville, UT). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Group Antibody Isotype Epitope Western dilution IHC dilution Source cdk5 cdk5 (C-8) Rabbit polyclonal cdk5 C terminus 1:100 1:100 Santa Cruz Biotechnology p35 p35 (C-19) Rabbit polyclonal p35 C terminus 1:100 nd Santa Cruz Biotechnology p67 p67 Rabbit polyclonal p67 N terminus 1:5000 nd H. Pant AP18 Mouse IgG1 MAP2B phosphorserine 136 1:1000 1:1000 L. Binder MAP2 AP20 Mouse IgG1 Total MAP2A, 2B and 2C 1:1000 1:500 Sigma R39 Rabbit polyclonal Total NF-H, NF-M and NF-L 1:2000 nd H. Pant SMI31 Mouse IgG1 Phospho-NF-H and NF-M 1:1000 1:500 Sternberger Monoclonals SMI34 Mouse IgG1 Phospho-NF-H 1:1000 nd Sternberger Monoclonals NFs SMI32 Mouse IgG1 Nonphosphorylated NF-H 1:500 1:500 Sternberger Monoclonals TG5 Mouse IgG1 Total tau 1:10 nd I. Vincent CP22 Mouse IgM Tau phosphothreonine 175 1:10 nd P. Davies CP13 Mouse IgG1 Tau phosphoserine 202 1:10 1:10 P. Davies MC6 Mouse IgG1 Tau phosphoserine 235 1:10 nd P. Davies MC15 Mouse IgM Tau phosphoserine 235 1:10 nd P. Davies Tau PHF1 Mouse IgG1 Tau phosphoserine 396/404 1:20 1:10 P. Davies Neuronal antigen NeuN Mouse IgG1 Neuronal nuclei 1:250 nd Chemicon Syntaxin SP6 Mouse IgG1 Syntaxin 1:20 1:10 W. Honer GSK3β 9332 Rabbit polyclonal Total GSK3β 1:500 nd Cell Signaling Technology β-Catenin 9562 Rabbit polyclonal Total β-catenin 1:500 nd Cell Signaling Technology Open in a separate window nd, Not determined.

Techniques: Incubation, Staining