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anti ndel1 antibody (Proteintech)

Name: anti ndel1 antibody
Brand: Proteintech
Rating: 93 (12 reviews)

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Proteintech anti ndel1 antibody
Anti Ndel1 Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 12 article reviews

anti ndel1 antibody - by Bioz Stars, 2026-02

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Identification of a de novo pathogenic NDEL1 variant in patients with pachygyria and subcortical heterotopia. a Brain MRI of the patients. In Case 1, the axial T1 image at 9 months of age shows diffuse, thick (> 10 mm) pachygyria, with the posterior regions more affected than the anterior regions. Sagittal view at 1 year and 8 months of age) shows a mildly thickened corpus callosum. The basal ganglion, brain stem, and cerebellum show no dysplasia or hypoplasia. In Case 2, the axial T1 imaging at 3 years old shows anterior pachygyria and posterior subcortical band heterotopia (double cortex). The sagittal T2 image shows a mildly thickened corpus callosum. b The bar graph shows the top ten genes prioritized by the Phenolyzer tool, ranking NDEL1 as the most important gene associated with lissencephaly. The X-axis represents the weighted score calculated by the Phenolyzer tool ( https://phenolyzer.wglab.org ) . HI: Haploinsufficiency Score; RVIS: Residual Variant Intolerance Score. NDEL1 was also ranked as the most important gene by the VarElect tool (data not shown). c The WES study identified the same de novo missense variant in NDEL1 : exon4: c.314G > C, p.R105P, in both patients with different mosaicism levels (25–30% in Case and 20–25% in Case 2). d Sanger sequencing of the patients and their parents confirms that the missense variant was de novo in origin. e Schematic representation of the relative location of p.R105P to known protein domains in NDEL1

Journal: Acta Neuropathologica

Article Title: Novel lissencephaly-associated NDEL1 variant reveals distinct roles of NDE1 and NDEL1 in nucleokinesis and human cortical malformations

doi: 10.1007/s00401-023-02665-y

Figure Lengend Snippet: Identification of a de novo pathogenic NDEL1 variant in patients with pachygyria and subcortical heterotopia. a Brain MRI of the patients. In Case 1, the axial T1 image at 9 months of age shows diffuse, thick (> 10 mm) pachygyria, with the posterior regions more affected than the anterior regions. Sagittal view at 1 year and 8 months of age) shows a mildly thickened corpus callosum. The basal ganglion, brain stem, and cerebellum show no dysplasia or hypoplasia. In Case 2, the axial T1 imaging at 3 years old shows anterior pachygyria and posterior subcortical band heterotopia (double cortex). The sagittal T2 image shows a mildly thickened corpus callosum. b The bar graph shows the top ten genes prioritized by the Phenolyzer tool, ranking NDEL1 as the most important gene associated with lissencephaly. The X-axis represents the weighted score calculated by the Phenolyzer tool ( https://phenolyzer.wglab.org ) . HI: Haploinsufficiency Score; RVIS: Residual Variant Intolerance Score. NDEL1 was also ranked as the most important gene by the VarElect tool (data not shown). c The WES study identified the same de novo missense variant in NDEL1 : exon4: c.314G > C, p.R105P, in both patients with different mosaicism levels (25–30% in Case and 20–25% in Case 2). d Sanger sequencing of the patients and their parents confirms that the missense variant was de novo in origin. e Schematic representation of the relative location of p.R105P to known protein domains in NDEL1

Article Snippet: The antibodies used and their concentrations were as follows: NDEL1(Rabbit polyclonal, Abcam, ab25959) 1:1000, NDE1 (Rabbit polyclonal, Proteintech, 10,233–1-AP) 1:1000, LIS1 (Goat polyclonal, Sigma-Aldrich, SAB2500597) 1:1000, Beta-actin (Mouse monoclonal, Proteintech, 66,009–1-Ig) 1:10,000, and Alpha tubulin (Mouse monoclonal, Proteintech, 66,031–1-Ig) 1:10,000.

Techniques: Variant Assay, Imaging, Sequencing

Expression of Nde1/NDE1 and Ndel1/NDEL1 in the developing mouse and human cortices. a UMAP plot of scRNA-seq of mouse cortex at E13.5 and E15.5 and human cortex at GW18. Three developmental states (progenitors, precursors, and neurons) are designated based on their marker genes (Supplementary Fig. 1). Heat maps of Nde1 / NDE1 , Ndel1 / NDEL1 , and Pafah1b1 / PAFAH1B1 ( Lis1 / LIS1 ) expression show specific expression patterns. b Violin plots of Nde1 / NDE1 , Ndel1 / NDEL1 , and Pafah1b1 / PAFAH1B1 ( Lis1 / LIS1 ) expression at three neuronal developmental states (top). The markers Pax6 / PAX6 , Eomes / EOMES , and Neurod6 / NEUROD6 are shown to distinguish these developmental stages. Bar graphs show the ratio of cells expressing Nde1 / NDE1 and Ndel1 / NDEL1 at each developmental state (bottom). Both plots show preferential expression of Nde1 / NDE1 in the progenitor state. c Violin plots of Nde1 / NDE1 , Ndel1 / NDEL1 , and Pafah1b1 / PAFAH1B1 ( Lis1 / LIS1 ) expression in progenitors at different cell cycle stages (top). Bar graphs show the expression of Nde1 / NDE1 and Ndel1 / NDEL1 at each cell cycle stage (bottom). Both plots show relatively high expression of Nde1 / NDE1 at the G2/M phase. d Gene expression profiles in mouse telencephalon at E15.5. Left: Spatial distribution and UMAP plot of individual capture dots grouped into 12 clusters based on their expression profiles. Right: The spatial distribution and violin plot of the expression of Nde1 , Ndel1 , and Lis1 in the dorsal telencephalon. While Nde1 is strongly expressed in the VZ, Ndel1 is mainly expressed in the IZ and CP. The color scale represents the log-normalization of the unique molecular identifier (UMI) count (also see Supplementary Fig. 2)

Journal: Acta Neuropathologica

Article Title: Novel lissencephaly-associated NDEL1 variant reveals distinct roles of NDE1 and NDEL1 in nucleokinesis and human cortical malformations

doi: 10.1007/s00401-023-02665-y

Figure Lengend Snippet: Expression of Nde1/NDE1 and Ndel1/NDEL1 in the developing mouse and human cortices. a UMAP plot of scRNA-seq of mouse cortex at E13.5 and E15.5 and human cortex at GW18. Three developmental states (progenitors, precursors, and neurons) are designated based on their marker genes (Supplementary Fig. 1). Heat maps of Nde1 / NDE1 , Ndel1 / NDEL1 , and Pafah1b1 / PAFAH1B1 ( Lis1 / LIS1 ) expression show specific expression patterns. b Violin plots of Nde1 / NDE1 , Ndel1 / NDEL1 , and Pafah1b1 / PAFAH1B1 ( Lis1 / LIS1 ) expression at three neuronal developmental states (top). The markers Pax6 / PAX6 , Eomes / EOMES , and Neurod6 / NEUROD6 are shown to distinguish these developmental stages. Bar graphs show the ratio of cells expressing Nde1 / NDE1 and Ndel1 / NDEL1 at each developmental state (bottom). Both plots show preferential expression of Nde1 / NDE1 in the progenitor state. c Violin plots of Nde1 / NDE1 , Ndel1 / NDEL1 , and Pafah1b1 / PAFAH1B1 ( Lis1 / LIS1 ) expression in progenitors at different cell cycle stages (top). Bar graphs show the expression of Nde1 / NDE1 and Ndel1 / NDEL1 at each cell cycle stage (bottom). Both plots show relatively high expression of Nde1 / NDE1 at the G2/M phase. d Gene expression profiles in mouse telencephalon at E15.5. Left: Spatial distribution and UMAP plot of individual capture dots grouped into 12 clusters based on their expression profiles. Right: The spatial distribution and violin plot of the expression of Nde1 , Ndel1 , and Lis1 in the dorsal telencephalon. While Nde1 is strongly expressed in the VZ, Ndel1 is mainly expressed in the IZ and CP. The color scale represents the log-normalization of the unique molecular identifier (UMI) count (also see Supplementary Fig. 2)

Techniques: Expressing, Marker

Effects of Ndel1 knockdown and p.R105P variant on neuronal distribution in embryonic mouse brains. a Western blot of NDEL1 protein in primary cultures at DIV 6 after infection with viruses encoding control or 4 different Ndel1 shRNA. Beta-actin was used as the loading control. The bar graph shows relative NDEL1 expression in these cells. Error bars represent SEM. One-way ANOVA; post hoc: Bonferroni test. *: p < 0.05, **: p < 0.01. b Coronal sections of E18.5 brains electroporated with shCtrl or sh Ndel1 along with GFP (green) at E14.5. While most GFP + cells reached the CP, cells expressing sh Ndel1 #16 were mainly restricted to the IZ and VZ/SVZ. This effect was rescued by the expression of Ndel1 WT but not the p.R105P variant. c Coronal sections of E18.5 brains electroporated with Ndel1 WT, p.R105P, p.R105Q, p.R105W, or the empty vector along with GFP (green) at E14.5. GFP + cells electroporated with pCAGIG-NDEL1, pCAGIG-R105Q, pCAGIG-R105W, pNeuroD1-NDEL1(WT), or vector were mainly redistributed to the CP. However, the expression of CAG / NeuroD1 promoter-driven p.R105P caused a nearly complete blockade of neurons from migrating to the CP. Slices were stained with DAPI (blue) to mark the nuclei of the cells. Bars = 100 μm. d The bar graph shows the percentage of GFP + cells in the CP, IZ, and VZ 4 days after electroporation in ( b ) and ( c ) ( n = 3 pregnant females in each condition). Error bars represent SEM. **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. One-way ANOVA; post hoc: Bonferroni test (color figure online)

Journal: Acta Neuropathologica

Article Title: Novel lissencephaly-associated NDEL1 variant reveals distinct roles of NDE1 and NDEL1 in nucleokinesis and human cortical malformations

doi: 10.1007/s00401-023-02665-y

Figure Lengend Snippet: Effects of Ndel1 knockdown and p.R105P variant on neuronal distribution in embryonic mouse brains. a Western blot of NDEL1 protein in primary cultures at DIV 6 after infection with viruses encoding control or 4 different Ndel1 shRNA. Beta-actin was used as the loading control. The bar graph shows relative NDEL1 expression in these cells. Error bars represent SEM. One-way ANOVA; post hoc: Bonferroni test. *: p < 0.05, **: p < 0.01. b Coronal sections of E18.5 brains electroporated with shCtrl or sh Ndel1 along with GFP (green) at E14.5. While most GFP + cells reached the CP, cells expressing sh Ndel1 #16 were mainly restricted to the IZ and VZ/SVZ. This effect was rescued by the expression of Ndel1 WT but not the p.R105P variant. c Coronal sections of E18.5 brains electroporated with Ndel1 WT, p.R105P, p.R105Q, p.R105W, or the empty vector along with GFP (green) at E14.5. GFP + cells electroporated with pCAGIG-NDEL1, pCAGIG-R105Q, pCAGIG-R105W, pNeuroD1-NDEL1(WT), or vector were mainly redistributed to the CP. However, the expression of CAG / NeuroD1 promoter-driven p.R105P caused a nearly complete blockade of neurons from migrating to the CP. Slices were stained with DAPI (blue) to mark the nuclei of the cells. Bars = 100 μm. d The bar graph shows the percentage of GFP + cells in the CP, IZ, and VZ 4 days after electroporation in ( b ) and ( c ) ( n = 3 pregnant females in each condition). Error bars represent SEM. **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. One-way ANOVA; post hoc: Bonferroni test (color figure online)

Techniques: Variant Assay, Western Blot, Infection, shRNA, Expressing, Plasmid Preparation, Staining, Electroporation

Expression of neuronal markers in cells expressing NDEL1 WT or p.R105P at P7. a Coronal sections at P7 from mouse brains electroporated with Ndel1 WT, p.R105Q, p.R105W, p.R105P, or the empty vector (green) at E14.5 were stained with layer II/III marker, BRN2 (red). Most of the cells electroporated with Ndel1 WT, p.R105Q, p.R105W, and p.R105P were BRN2 + , similar to the control cells electroporated with the empty vector, even though p.R105P-expressing cells were arrested in the WM. b Similarly, in brains electroporated with Ndel1 WT, p.R105Q, p.R105W, p.R105P, and the empty vector, most of the electroporated cells were NeuN + (red). The regions within the white boxes were magnified. Bars = 200 μm in the wide field images and 20 μm in high magnification images. Bar graphs show the percentage of BRN2 + /GFP + and NeuN + /GFP + cells in the electroporated brain slices ( n = 3 mice from 3 independent pregnancies). Error bars represent SEM. One-way ANOVA; post hoc: Bonferroni test. ns: not significant (color figure online)

Journal: Acta Neuropathologica

Article Title: Novel lissencephaly-associated NDEL1 variant reveals distinct roles of NDE1 and NDEL1 in nucleokinesis and human cortical malformations

doi: 10.1007/s00401-023-02665-y

Figure Lengend Snippet: Expression of neuronal markers in cells expressing NDEL1 WT or p.R105P at P7. a Coronal sections at P7 from mouse brains electroporated with Ndel1 WT, p.R105Q, p.R105W, p.R105P, or the empty vector (green) at E14.5 were stained with layer II/III marker, BRN2 (red). Most of the cells electroporated with Ndel1 WT, p.R105Q, p.R105W, and p.R105P were BRN2 + , similar to the control cells electroporated with the empty vector, even though p.R105P-expressing cells were arrested in the WM. b Similarly, in brains electroporated with Ndel1 WT, p.R105Q, p.R105W, p.R105P, and the empty vector, most of the electroporated cells were NeuN + (red). The regions within the white boxes were magnified. Bars = 200 μm in the wide field images and 20 μm in high magnification images. Bar graphs show the percentage of BRN2 + /GFP + and NeuN + /GFP + cells in the electroporated brain slices ( n = 3 mice from 3 independent pregnancies). Error bars represent SEM. One-way ANOVA; post hoc: Bonferroni test. ns: not significant (color figure online)

Techniques: Expressing, Plasmid Preparation, Staining, Marker

NDEL1 p.R105P increased the length of the leading process and the nucleus–centrosome distance (N–C distance). a Representative images of GFP + neurons in E18.5 mouse brain electroporated with empty vector, Ndel1 WT or p.R105P cDNA along with GFP (green) at E14.5. Arrows represent the leading process from the cell body. Bars = 20 μm. Cells expressing p.R105P extended a very long leading process compared to those cells electroporated with the empty vector or Ndel1 WT. The spot graph shows the length of the leading process in cells electroporated with the empty vector ( n = 92 cells, acquired from 3 mice), Ndel1 WT ( n = 138 cells, acquired from 3 mice), or p.R105P ( n = 140 cells, acquired from 3 mice). Error bars represent SEM. ****: p < 0.0001. Kruskal–Wallis test; post hoc: Dunn’s test. ns: not significant. b Representative images of migrating neurons electroporated with Ndel1 WT or p.R105P cDNA along with GFP (green) and CentrinII-DsRed (red). The brains were electroporated at E14.5 and harvested at E17.5. The centrosomes (arrowhead) in most cells expressing empty vector and Ndel1 WT were found in the perinuclear area, while the centrosomes in cells expressing p.R105P were often found further along the leading process at a longer distance from the nucleus (dashed lines). Spot graph shows the N–C distance in cells electroporated with empty vector ( n = 130 cells, acquired from 3 mice), Ndel1 WT ( n = 91 cells, acquired from 3 mice), or p.R105P ( n = 77 cells, acquired from 3 mice). Error bars represent SEM. ****: p < 0.0001. Kruskal–Wallis test; post-hoc: Dunn’s test. ns: not significant (color figure online)

Journal: Acta Neuropathologica

Article Title: Novel lissencephaly-associated NDEL1 variant reveals distinct roles of NDE1 and NDEL1 in nucleokinesis and human cortical malformations

doi: 10.1007/s00401-023-02665-y

Figure Lengend Snippet: NDEL1 p.R105P increased the length of the leading process and the nucleus–centrosome distance (N–C distance). a Representative images of GFP + neurons in E18.5 mouse brain electroporated with empty vector, Ndel1 WT or p.R105P cDNA along with GFP (green) at E14.5. Arrows represent the leading process from the cell body. Bars = 20 μm. Cells expressing p.R105P extended a very long leading process compared to those cells electroporated with the empty vector or Ndel1 WT. The spot graph shows the length of the leading process in cells electroporated with the empty vector ( n = 92 cells, acquired from 3 mice), Ndel1 WT ( n = 138 cells, acquired from 3 mice), or p.R105P ( n = 140 cells, acquired from 3 mice). Error bars represent SEM. ****: p < 0.0001. Kruskal–Wallis test; post hoc: Dunn’s test. ns: not significant. b Representative images of migrating neurons electroporated with Ndel1 WT or p.R105P cDNA along with GFP (green) and CentrinII-DsRed (red). The brains were electroporated at E14.5 and harvested at E17.5. The centrosomes (arrowhead) in most cells expressing empty vector and Ndel1 WT were found in the perinuclear area, while the centrosomes in cells expressing p.R105P were often found further along the leading process at a longer distance from the nucleus (dashed lines). Spot graph shows the N–C distance in cells electroporated with empty vector ( n = 130 cells, acquired from 3 mice), Ndel1 WT ( n = 91 cells, acquired from 3 mice), or p.R105P ( n = 77 cells, acquired from 3 mice). Error bars represent SEM. ****: p < 0.0001. Kruskal–Wallis test; post-hoc: Dunn’s test. ns: not significant (color figure online)

Techniques: Plasmid Preparation, Expressing

The p.R105P variant disrupted NDEL1 interaction with LIS1 and decreased the localization at the centrosome. a Immunoprecipitation of NDEL1 WT and p.R105P to LIS1. Lysates from HEK293T cells transfected with Ndel1 WT, p.R105Q, p.R105W, or p.R105P for 24 h were immunoprecipitated by GFP-antibody. Western blots show that LIS1 co-precipitated with NDEL1 WT, p.R105Q, and p.R105W, but not the p.R105P variant. The bar graph shows co-precipitated LIS1 with p.R105Q ( n = 5), p.R105W ( n = 5), and p.R105P ( n = 8) relative to NDEL1 WT ( n = 8). Error bars represent SEM. **: p < 0.01, Kruskal–Wallis test; post hoc: Dunn’s test. Data of each repeat in each group were acquired from independent batches of experiments. b Centrosomal localization of NDEL1 WT and the p.R105P variant. U2OS cells were transfected with pEGFP-mNDEL1(WT) or pEGFP-mNDEL1(R105P) (green) for 24 h and stained with the centrosome marker CentrinII (red). The regions within the box were magnified. Bar = 5 μm. The spot graph shows the relative intensity of NDEL1 fluorescence at the centrosome to the cytosol. There was a dramatic decrease in the localization of p.R105P ( n = 138 cells) at the centrosome compared to WT ( n = 114 cells). Error bars represent SEM. ****: p < 0.0001. Mann–Whitney test. Data from each group were acquired from three independent batches of experiments (color figure online)

Journal: Acta Neuropathologica

Article Title: Novel lissencephaly-associated NDEL1 variant reveals distinct roles of NDE1 and NDEL1 in nucleokinesis and human cortical malformations

doi: 10.1007/s00401-023-02665-y

Figure Lengend Snippet: The p.R105P variant disrupted NDEL1 interaction with LIS1 and decreased the localization at the centrosome. a Immunoprecipitation of NDEL1 WT and p.R105P to LIS1. Lysates from HEK293T cells transfected with Ndel1 WT, p.R105Q, p.R105W, or p.R105P for 24 h were immunoprecipitated by GFP-antibody. Western blots show that LIS1 co-precipitated with NDEL1 WT, p.R105Q, and p.R105W, but not the p.R105P variant. The bar graph shows co-precipitated LIS1 with p.R105Q ( n = 5), p.R105W ( n = 5), and p.R105P ( n = 8) relative to NDEL1 WT ( n = 8). Error bars represent SEM. **: p < 0.01, Kruskal–Wallis test; post hoc: Dunn’s test. Data of each repeat in each group were acquired from independent batches of experiments. b Centrosomal localization of NDEL1 WT and the p.R105P variant. U2OS cells were transfected with pEGFP-mNDEL1(WT) or pEGFP-mNDEL1(R105P) (green) for 24 h and stained with the centrosome marker CentrinII (red). The regions within the box were magnified. Bar = 5 μm. The spot graph shows the relative intensity of NDEL1 fluorescence at the centrosome to the cytosol. There was a dramatic decrease in the localization of p.R105P ( n = 138 cells) at the centrosome compared to WT ( n = 114 cells). Error bars represent SEM. ****: p < 0.0001. Mann–Whitney test. Data from each group were acquired from three independent batches of experiments (color figure online)

Techniques: Variant Assay, Immunoprecipitation, Transfection, Western Blot, Staining, Marker, Fluorescence, MANN-WHITNEY

Schematic diagram depicting the distinct roles of NDE1 and NDEL1 in cortical development and the cellular and molecular mechanisms of NDEL1 p.R105P variant in causing lissencephaly. During cortical development, the nucleus of RGCs undergoes cell-cycle-dependent INM during proliferation to self-renew and produce neurons. Cytoplasmic dynein, supported by NDE1 and LIS1, facilitates downward movement, while KIF1A is responsible for upward movement. Dysfunction of NDE1 blocks downward movement and subsequent proliferation; its mutations lead to microcephaly in humans. Post-mitotic neurons migrate along the radial fibers of RGCs to populate the CP, where they become pyramidal neurons. During the migration process, NDEL1 interacts with both cytoplasmic dynein and LIS1 to regulate dynein motor activities, enabling nucleokinesis during neuronal migration. The p.R105P variant disrupts the interaction of NDEL1 with LIS1, which impairs the function of the cytoplasmic dynein complex. This defect impedes N–C coupling, blocks nucleokinesis, and impairs neuronal migration during cortical development. These defects, in turn, lead to the pathogenesis of human lissencephaly

Journal: Acta Neuropathologica

Article Title: Novel lissencephaly-associated NDEL1 variant reveals distinct roles of NDE1 and NDEL1 in nucleokinesis and human cortical malformations

doi: 10.1007/s00401-023-02665-y

Figure Lengend Snippet: Schematic diagram depicting the distinct roles of NDE1 and NDEL1 in cortical development and the cellular and molecular mechanisms of NDEL1 p.R105P variant in causing lissencephaly. During cortical development, the nucleus of RGCs undergoes cell-cycle-dependent INM during proliferation to self-renew and produce neurons. Cytoplasmic dynein, supported by NDE1 and LIS1, facilitates downward movement, while KIF1A is responsible for upward movement. Dysfunction of NDE1 blocks downward movement and subsequent proliferation; its mutations lead to microcephaly in humans. Post-mitotic neurons migrate along the radial fibers of RGCs to populate the CP, where they become pyramidal neurons. During the migration process, NDEL1 interacts with both cytoplasmic dynein and LIS1 to regulate dynein motor activities, enabling nucleokinesis during neuronal migration. The p.R105P variant disrupts the interaction of NDEL1 with LIS1, which impairs the function of the cytoplasmic dynein complex. This defect impedes N–C coupling, blocks nucleokinesis, and impairs neuronal migration during cortical development. These defects, in turn, lead to the pathogenesis of human lissencephaly

Techniques: Variant Assay, Migration

$(A) Dynamin2 (Dyn2) co-immunoprecipitates with Ndel1, so do Lis1 and Dynein intermediate chain (DIC) in HeLa cells. Co-immunoprecipitations in absence of antibody (noAb) or with Myc antibody serve as negative controls. (B) Dyn2 co-immunoprecipitates with Ndel1 in diverse cell types (HeLa cells, neuroblastoma CAD cells, primary cultured rat hippocampal neurons) and mouse cortex. (C) Co-immunoprecipitation of Dyn2 with Ndel1 in a post-mitochondrial fraction from HeLa cells (left panel). Co-immunoprecipitations in absence of antibody (No Abs) or with Myc antibody serve as negative controls. The right panel presents a Western blot analysis of the membranes content of a post-mitochondrial fraction. Endoplasmic reticulum (ER), trans-Golgi (TGN), plasma membrane, endosomes and cytosolic proteins are all present in this fraction, as detected by KDEL ER marker/BiP, p230 trans-Golgi, Na + /K + ATPase, EEA1 and Tubulin antibodies, respectively. (D) In vitro pull-down of His-Ndel1 by GST-Dyn2 but not GST. The protein detected by anti-GST antibodies in GST-Dyn2 pull-down corresponds to the cleaved GST from GST-Dyn2. (E) Far-western assays demonstrating the direct interaction between Ndel1 and Dyn2. His-Ndel1 bound to membranes was overlaid with GST-Dyn2 protein that was detected with a Dyn2 antibody at the His-Ndel1 molecular weight (∼45 kDa). Consistently, GST-Dyn2 bound membranes overlaid with His-Ndel1 that was detected with a Ndel1 antibody at the GST-Dyn2 molecular weight (∼125 kDa = ∼100 kDa for Dyn2+∼25 kDa for GST).$

Journal: PLoS ONE

Article Title: The Cytoskeletal Protein Ndel1 Regulates Dynamin 2 GTPase Activity

doi: 10.1371/journal.pone.0014583

Figure Lengend Snippet: (A) Dynamin2 (Dyn2) co-immunoprecipitates with Ndel1, so do Lis1 and Dynein intermediate chain (DIC) in HeLa cells. Co-immunoprecipitations in absence of antibody (noAb) or with Myc antibody serve as negative controls. (B) Dyn2 co-immunoprecipitates with Ndel1 in diverse cell types (HeLa cells, neuroblastoma CAD cells, primary cultured rat hippocampal neurons) and mouse cortex. (C) Co-immunoprecipitation of Dyn2 with Ndel1 in a post-mitochondrial fraction from HeLa cells (left panel). Co-immunoprecipitations in absence of antibody (No Abs) or with Myc antibody serve as negative controls. The right panel presents a Western blot analysis of the membranes content of a post-mitochondrial fraction. Endoplasmic reticulum (ER), trans-Golgi (TGN), plasma membrane, endosomes and cytosolic proteins are all present in this fraction, as detected by KDEL ER marker/BiP, p230 trans-Golgi, Na + /K + ATPase, EEA1 and Tubulin antibodies, respectively. (D) In vitro pull-down of His-Ndel1 by GST-Dyn2 but not GST. The protein detected by anti-GST antibodies in GST-Dyn2 pull-down corresponds to the cleaved GST from GST-Dyn2. (E) Far-western assays demonstrating the direct interaction between Ndel1 and Dyn2. His-Ndel1 bound to membranes was overlaid with GST-Dyn2 protein that was detected with a Dyn2 antibody at the His-Ndel1 molecular weight (∼45 kDa). Consistently, GST-Dyn2 bound membranes overlaid with His-Ndel1 that was detected with a Ndel1 antibody at the GST-Dyn2 molecular weight (∼125 kDa = ∼100 kDa for Dyn2+∼25 kDa for GST).

Article Snippet: GST-fused Dyn2 ( M r of ∼130 kDa) and His-tagged Ndel1 ( M r of ∼45 kDa) proteins were expressed in BL-21 Escherichia coli (Stratagene) cells and purified on a Glutathione Sepharose 4 Fast Flow column (GE Healthcare) or Ni-NTA Agarose column (Qiagen) according to the manufacturer's protocol.

Techniques: Cell Culture, Immunoprecipitation, Western Blot, Marker, In Vitro, Molecular Weight

(A) GST-tagged constructs of Dyn2 used for GST pull-downs. Mid, middle domain; PH, pleckstrin homology domain; GED, GTPase effector domain; PRD, proline-rich domain. (B) GST pull-downs experiments indicating that all domains in Dyn2 with the exception of the PRD bind to His-Ndel1. (C) Endogenous Dyn2 co-immunoprecipitates with Ndel1 full length (fl) (a.a. 1–345) and C-terminus (tail) (a.a. 191–345) in transfected HeLa cells. *Bands that have been confirmed with Ndel1 antibodies. C, cells transfected with an empty vector. (D) Dyn2 full length interacts with Ndel1 full length (a.a. 1–345) and Ndel1 tail (a.a. 191–345) but not Ndel1 coiled-coil domain (a.a. 1–201) in yeast 2-hybrid assays as detected by X-Gal and 3-AT.

Journal: PLoS ONE

Article Title: The Cytoskeletal Protein Ndel1 Regulates Dynamin 2 GTPase Activity

doi: 10.1371/journal.pone.0014583

Figure Lengend Snippet: (A) GST-tagged constructs of Dyn2 used for GST pull-downs. Mid, middle domain; PH, pleckstrin homology domain; GED, GTPase effector domain; PRD, proline-rich domain. (B) GST pull-downs experiments indicating that all domains in Dyn2 with the exception of the PRD bind to His-Ndel1. (C) Endogenous Dyn2 co-immunoprecipitates with Ndel1 full length (fl) (a.a. 1–345) and C-terminus (tail) (a.a. 191–345) in transfected HeLa cells. *Bands that have been confirmed with Ndel1 antibodies. C, cells transfected with an empty vector. (D) Dyn2 full length interacts with Ndel1 full length (a.a. 1–345) and Ndel1 tail (a.a. 191–345) but not Ndel1 coiled-coil domain (a.a. 1–201) in yeast 2-hybrid assays as detected by X-Gal and 3-AT.

Techniques: Construct, Transfection, Plasmid Preparation

(A) Radioactive GTPase assays for Ndel1 (red), Dyn2 full length (FL, orange) and Ndel1 together with Dyn2 FL (green). Ndel1 itself does not have intrinsic GTPase activity. The addition of Ndel1 to oligomerized Dyn2 FL enhances the activity of the latter by ∼2 fold over 60 minutes. Error bars indicate s.d. (n = 4). Two-way ANOVA: p<0.0001. (B) Non-radioactive GTPase assays for Dyn2 FL. Ndel1 does not show detectable GTPase activity, but increases the GTPase activity of assembled Dyn2 FL. The activator Phospholipase D (PLD, positive control) also increases the activity of oligomerized Dyn2 FL. Under high salt conditions (negative control), unassembled Dyn2 FL shows reduced GTPase activity when compared to assembled Dyn2 FL. The presence of Ndel1 enhances Dyn2 FL GTPase activity even under high salt conditions. Error bars indicate S.E.M. (n = 3). Two-way ANOVA: p<0.0001 for all conditions. (C) Non-radioactive GTPase assay for F1 and F5. Ndel1 itself does not have detectable intrinsic GTPase activity. The addition of Ndel1 increases the GTPase activity of oligomeric F5. PLD and high salt conditions elevates and diminishes F5 GTPase activity, respectively. The presence of Ndel1 enhances F5 activity even under high salt conditions. Note that the GTPase activity of F1, which cannot forms oligomers, is below detectable levels but becomes recordable following addition of Ndel1 or PLD. Error bars indicate S.E.M. (n = 3). Two-way ANOVA: p<0.0005 for all conditions.

Journal: PLoS ONE

Article Title: The Cytoskeletal Protein Ndel1 Regulates Dynamin 2 GTPase Activity

doi: 10.1371/journal.pone.0014583

Figure Lengend Snippet: (A) Radioactive GTPase assays for Ndel1 (red), Dyn2 full length (FL, orange) and Ndel1 together with Dyn2 FL (green). Ndel1 itself does not have intrinsic GTPase activity. The addition of Ndel1 to oligomerized Dyn2 FL enhances the activity of the latter by ∼2 fold over 60 minutes. Error bars indicate s.d. (n = 4). Two-way ANOVA: p<0.0001. (B) Non-radioactive GTPase assays for Dyn2 FL. Ndel1 does not show detectable GTPase activity, but increases the GTPase activity of assembled Dyn2 FL. The activator Phospholipase D (PLD, positive control) also increases the activity of oligomerized Dyn2 FL. Under high salt conditions (negative control), unassembled Dyn2 FL shows reduced GTPase activity when compared to assembled Dyn2 FL. The presence of Ndel1 enhances Dyn2 FL GTPase activity even under high salt conditions. Error bars indicate S.E.M. (n = 3). Two-way ANOVA: p<0.0001 for all conditions. (C) Non-radioactive GTPase assay for F1 and F5. Ndel1 itself does not have detectable intrinsic GTPase activity. The addition of Ndel1 increases the GTPase activity of oligomeric F5. PLD and high salt conditions elevates and diminishes F5 GTPase activity, respectively. The presence of Ndel1 enhances F5 activity even under high salt conditions. Note that the GTPase activity of F1, which cannot forms oligomers, is below detectable levels but becomes recordable following addition of Ndel1 or PLD. Error bars indicate S.E.M. (n = 3). Two-way ANOVA: p<0.0005 for all conditions.

Techniques: Activity Assay, Positive Control, Negative Control

A sedimentation assay for Dyn2 with or without Ndel1 detects lower amounts of Dyn2 in pellet of samples with Ndel1 protein vs without Ndel1 protein. Error bars indicate s.d. (n = 3). Student t -test: *, p<0.05.

Journal: PLoS ONE

Article Title: The Cytoskeletal Protein Ndel1 Regulates Dynamin 2 GTPase Activity

doi: 10.1371/journal.pone.0014583

Figure Lengend Snippet: A sedimentation assay for Dyn2 with or without Ndel1 detects lower amounts of Dyn2 in pellet of samples with Ndel1 protein vs without Ndel1 protein. Error bars indicate s.d. (n = 3). Student t -test: *, p<0.05.

Techniques: Sedimentation

$Fractionation of GluR1 in heavy membranes (HM) and light membranes (LM) from HeLa cells transfected with a combination of Dyn2, GluR1, Flag-Ndel1, Dyn2(K44A) constructs and/or treated with Ndel1 siRNA. (A) The HM fraction comprises several organelles, including the trans-Golgi network (TGN) and the endoplasmic reticulum (ER), as detected with p230 trans-Golgi/TGN38 and KDEL ER marker antibodies, respectively. The LM fraction includes cytosolic proteins and small organelles like early endosomes as indicated by Tubulin and EEA1 antibodies, respectively. (B) The framed Western blots depict the separation of GluR1 in HM vs GluR1 in LM in HeLa cells. Increasing the levels of Dyn2 or Ndel1 reduces the ratio GluR1 HM/GluR1 (HM+LM), indicating GluR1 is redistributed from the heavy membranes to the lighter membranes. The usage of a dominant negative mutant of Dyn2 (Dyn2(K44A)) or the treatment of cells with Ndel1 siRNA reverts the ratio, indicating accumulating GluR1 in the HM fraction. “C” corresponds to control (cells transfected with an empty vector). Error bars indicate s.d. (n = 3). One way ANOVA: ***, p<0.001; **, p<0.01; *, p<0.05. Note that the levels of stable (acetylated) Tubulin are similar in Ndel1 siRNA-treated cells vs control siRNA-treated cells. (C) In HeLa cells treated with control siRNA, the AMPA receptor GluR1 (red) is found at the cell periphery and up to the cell edge. In HeLa cells treated with Ndel1 siRNA most of GluR1 (red) is found close to the nucleus with very little amount at the cell periphery. Cells were double stained with a marker for the trans-Golgi network (p230 trans-Golgi, blue). Scale bar, 10 µm.$

Journal: PLoS ONE

Article Title: The Cytoskeletal Protein Ndel1 Regulates Dynamin 2 GTPase Activity

doi: 10.1371/journal.pone.0014583

Figure Lengend Snippet: Fractionation of GluR1 in heavy membranes (HM) and light membranes (LM) from HeLa cells transfected with a combination of Dyn2, GluR1, Flag-Ndel1, Dyn2(K44A) constructs and/or treated with Ndel1 siRNA. (A) The HM fraction comprises several organelles, including the trans-Golgi network (TGN) and the endoplasmic reticulum (ER), as detected with p230 trans-Golgi/TGN38 and KDEL ER marker antibodies, respectively. The LM fraction includes cytosolic proteins and small organelles like early endosomes as indicated by Tubulin and EEA1 antibodies, respectively. (B) The framed Western blots depict the separation of GluR1 in HM vs GluR1 in LM in HeLa cells. Increasing the levels of Dyn2 or Ndel1 reduces the ratio GluR1 HM/GluR1 (HM+LM), indicating GluR1 is redistributed from the heavy membranes to the lighter membranes. The usage of a dominant negative mutant of Dyn2 (Dyn2(K44A)) or the treatment of cells with Ndel1 siRNA reverts the ratio, indicating accumulating GluR1 in the HM fraction. “C” corresponds to control (cells transfected with an empty vector). Error bars indicate s.d. (n = 3). One way ANOVA: ***, p<0.001; **, p<0.01; *, p<0.05. Note that the levels of stable (acetylated) Tubulin are similar in Ndel1 siRNA-treated cells vs control siRNA-treated cells. (C) In HeLa cells treated with control siRNA, the AMPA receptor GluR1 (red) is found at the cell periphery and up to the cell edge. In HeLa cells treated with Ndel1 siRNA most of GluR1 (red) is found close to the nucleus with very little amount at the cell periphery. Cells were double stained with a marker for the trans-Golgi network (p230 trans-Golgi, blue). Scale bar, 10 µm.

Techniques: Fractionation, Transfection, Construct, Marker, Western Blot, Dominant Negative Mutation, Plasmid Preparation, Staining

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