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TaKaRa control vector expressing gfp protein
Decreased dendritic complexity in <t>DSCAM-overexpressing</t> hippocampal neurons. A , Representative micrographs of hippocampal neurons transfected at DIV7 with the indicated constructs. Neuronal morphology was visualized by <t>GFP</t> immunocytochemistry 2 d after transfection. Dendrites were identified as MAP2-positive neurites. Scale bars, 60 μm. B , Sholl analysis of the neurons transfected with GFP or DSCAM-IRES-GFP vectors. C , Total dendritic length of neurons transfected with the indicated constructs. D , Number of primary dendrites of neurons transfected with the indicated constructs. E , Somatic area of neurons transfected with the indicated plasmids. Mean values are shown (12–15 neurons); the error bars indicate the SEM, and asterisks denote statistically significant differences: * p
Control Vector Expressing Gfp Protein, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Related Products / Commonly Used Together

fluorescent protein gfp protein
mouse dscam

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1) Product Images from "NMDA-Mediated Regulation of DSCAM Dendritic Local Translation Is Lost in a Mouse Model of Down's Syndrome"

Article Title: NMDA-Mediated Regulation of DSCAM Dendritic Local Translation Is Lost in a Mouse Model of Down's Syndrome

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.3457-10.2010

Decreased dendritic complexity in DSCAM-overexpressing hippocampal neurons. A , Representative micrographs of hippocampal neurons transfected at DIV7 with the indicated constructs. Neuronal morphology was visualized by GFP immunocytochemistry 2 d after transfection. Dendrites were identified as MAP2-positive neurites. Scale bars, 60 μm. B , Sholl analysis of the neurons transfected with GFP or DSCAM-IRES-GFP vectors. C , Total dendritic length of neurons transfected with the indicated constructs. D , Number of primary dendrites of neurons transfected with the indicated constructs. E , Somatic area of neurons transfected with the indicated plasmids. Mean values are shown (12–15 neurons); the error bars indicate the SEM, and asterisks denote statistically significant differences: * p
Figure Legend Snippet: Decreased dendritic complexity in DSCAM-overexpressing hippocampal neurons. A , Representative micrographs of hippocampal neurons transfected at DIV7 with the indicated constructs. Neuronal morphology was visualized by GFP immunocytochemistry 2 d after transfection. Dendrites were identified as MAP2-positive neurites. Scale bars, 60 μm. B , Sholl analysis of the neurons transfected with GFP or DSCAM-IRES-GFP vectors. C , Total dendritic length of neurons transfected with the indicated constructs. D , Number of primary dendrites of neurons transfected with the indicated constructs. E , Somatic area of neurons transfected with the indicated plasmids. Mean values are shown (12–15 neurons); the error bars indicate the SEM, and asterisks denote statistically significant differences: * p

Techniques Used: Transfection, Construct, Immunocytochemistry

Related Articles

Transfection:

Article Title: NMDA-Mediated Regulation of DSCAM Dendritic Local Translation Is Lost in a Mouse Model of Down's Syndrome
Article Snippet: .. A total of 0.8 μg of a plasmid expressing mouse DSCAM along with green fluorescent protein (GFP) protein (CMV-DSCAM-IRES-GFP) ( ) or a control vector expressing GFP protein (pEGFP-C1; Clontech) was transfected per well. .. Effects on dendrite arborization were analyzed 2 d after transfection.

Plasmid Preparation:

Article Title: NMDA-Mediated Regulation of DSCAM Dendritic Local Translation Is Lost in a Mouse Model of Down's Syndrome
Article Snippet: .. A total of 0.8 μg of a plasmid expressing mouse DSCAM along with green fluorescent protein (GFP) protein (CMV-DSCAM-IRES-GFP) ( ) or a control vector expressing GFP protein (pEGFP-C1; Clontech) was transfected per well. .. Effects on dendrite arborization were analyzed 2 d after transfection.

Expressing:

Article Title: NMDA-Mediated Regulation of DSCAM Dendritic Local Translation Is Lost in a Mouse Model of Down's Syndrome
Article Snippet: .. A total of 0.8 μg of a plasmid expressing mouse DSCAM along with green fluorescent protein (GFP) protein (CMV-DSCAM-IRES-GFP) ( ) or a control vector expressing GFP protein (pEGFP-C1; Clontech) was transfected per well. .. Effects on dendrite arborization were analyzed 2 d after transfection.

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    TaKaRa gfp encoding mammalian expression vector
    S655 phosphorylation is a targeting signal for retrieval of <t>APP</t> to the Golgi . (A) Wt, S655A and S655E <t>APP-GFP</t> at the cell surface were labelled (0 min) by incubating COS-7 cells with the 22C11 anti-APP ectodomain antibody (
    Gfp Encoding Mammalian Expression Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    TaKaRa pegfp n1
    Apoptosis assay with CHO cells expressing scFv-caspase and specific antigen. ( A ) CHO cells were cotransfected with <t>pEGFP-N1</t> and clones encoding various scFv fusions. Transfected cells were identified by fluorescence and sorted with a FACSCalibur cell sorter. Genomic DNA was extracted from selected cells and PCR amplifications carried out with the ApoAlert LM-PCR Ladder Assay Kit. The products were fractionated on 1.5% agarose. The lanes correspond to PCRs from DNA of cells that had been transfected with plasmids expressing scFvR4-VP16 + β-gal (lane 1), scFvR4-caspase 3 + β-gal (lane 2), scFvR4-caspase 3(C163S) + β-gal (lane 3), scFvF8-caspase 3 + β-gal (lane 4), or scFvR4-caspase 3 alone (lane 5). The negative PCR control represents a product from a reaction identical with the others but lacking template DNA. ( B ) PCRs were performed with the same DNA samples as in A , but using primers specific for the Chinese hamster actin gene. The negative control was as in A ; the positive control was the reaction product obtained from a PCR amplification with purified CHO genomic DNA.
    Pegfp N1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 1547 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1/product/TaKaRa
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    TaKaRa control vector expressing gfp protein
    Decreased dendritic complexity in <t>DSCAM-overexpressing</t> hippocampal neurons. A , Representative micrographs of hippocampal neurons transfected at DIV7 with the indicated constructs. Neuronal morphology was visualized by <t>GFP</t> immunocytochemistry 2 d after transfection. Dendrites were identified as MAP2-positive neurites. Scale bars, 60 μm. B , Sholl analysis of the neurons transfected with GFP or DSCAM-IRES-GFP vectors. C , Total dendritic length of neurons transfected with the indicated constructs. D , Number of primary dendrites of neurons transfected with the indicated constructs. E , Somatic area of neurons transfected with the indicated plasmids. Mean values are shown (12–15 neurons); the error bars indicate the SEM, and asterisks denote statistically significant differences: * p
    Control Vector Expressing Gfp Protein, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/control vector expressing gfp protein/product/TaKaRa
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    control vector expressing gfp protein - by Bioz Stars, 2020-08
    85/100 stars
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    80
    TaKaRa control green fluorescent protein gfp expression vector
    Decreased dendritic complexity in <t>DSCAM-overexpressing</t> hippocampal neurons. A , Representative micrographs of hippocampal neurons transfected at DIV7 with the indicated constructs. Neuronal morphology was visualized by <t>GFP</t> immunocytochemistry 2 d after transfection. Dendrites were identified as MAP2-positive neurites. Scale bars, 60 μm. B , Sholl analysis of the neurons transfected with GFP or DSCAM-IRES-GFP vectors. C , Total dendritic length of neurons transfected with the indicated constructs. D , Number of primary dendrites of neurons transfected with the indicated constructs. E , Somatic area of neurons transfected with the indicated plasmids. Mean values are shown (12–15 neurons); the error bars indicate the SEM, and asterisks denote statistically significant differences: * p
    Control Green Fluorescent Protein Gfp Expression Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/control green fluorescent protein gfp expression vector/product/TaKaRa
    Average 80 stars, based on 1 article reviews
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    control green fluorescent protein gfp expression vector - by Bioz Stars, 2020-08
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    Image Search Results


    S655 phosphorylation is a targeting signal for retrieval of APP to the Golgi . (A) Wt, S655A and S655E APP-GFP at the cell surface were labelled (0 min) by incubating COS-7 cells with the 22C11 anti-APP ectodomain antibody (

    Journal: Molecular Neurodegeneration

    Article Title: Retrieval of the Alzheimer's amyloid precursor protein from the endosome to the TGN is S655 phosphorylation state-dependent and retromer-mediated

    doi: 10.1186/1750-1326-5-40

    Figure Lengend Snippet: S655 phosphorylation is a targeting signal for retrieval of APP to the Golgi . (A) Wt, S655A and S655E APP-GFP at the cell surface were labelled (0 min) by incubating COS-7 cells with the 22C11 anti-APP ectodomain antibody ("22C11 Uptake", Texas red staining) at 4°C. Following 15 and 30 min of incubation at 37°C, the localization of APP-GFP endocytic vesicles (green/red-22C11 co-localizing vesicles) were monitored. Arrows indicate the Golgi area and APP-GFP/22C11 endocytic vesicles co-localizing to or around the same region. ROI - region of interest denoting the Golgi area and co-localizing vesicles. (B) The highly green-fluorescent perinuclear structure in APP-GFP transfected cells was confirmed to be the Golgi in Wt APP-GFP and CFP-Golgi co-transfected cells, upon 30 min of 22C11 uptake. Bar, 10 μm. (C) confocal quantitative analysis of the degree of co-localization between uptaken 22C11 and the APP-GPP population at the Golgi area at 30 min of 22C11 antibody uptake (AU) at 37°C. Values are mean ± SEM, n = 20 cells. Statistical significance symbols used were: (*) for comparison of S655 phosphomutant and Wt data; ( + ) S655A vs S655E data. Statistical significance levels are presented as (**), for p

    Article Snippet: The resultant fragments were digested with endonucleases (Age I and Nru I) and subcloned into the Age I/Sma I restriction sites of the GFP-encoding mammalian expression vector (pEGFP-N1, Clontech) as N-terminal APP-GFP translational fusions.

    Techniques: Staining, Incubation, Transfection

    VPS35 down-regulation impairs S655E APP-GFP retrieval to the TGN and decreases its turnover rate . (A) Left: Immunodetection of VPS35 levels in COS-7 cells co-transfected with N1 EGFP and several concentrations of anti-VPS35 siRNAs for 24 h. Tubulin was probed as a control. Right: Quantification of VPS35 levels, plotted as percentages of control conditions (cells only transfected with N1 EGFP). Values are mean ± SEM (n = 2-6); (#), statistical significance of p ≤ 0.05 for siRNA VPS35 vs control data. (B) The 15 min 22C11 antibody uptake assay (Alexa350 blue staining) was applied to monitor S655E APP-GFP protein retrieval from the endosome to the TGN upon COS-7 cells pre-incubation with 5nM VPS35 siRNAs for 24 h to downregulate VPS35 (Texas red staining). (B.I) Control: cells only transfected with S655E APP-GFP. (B.II - B.III) siRNA VPS35: cells co-transfected with S655E APP-GFP and VPS35 siRNAs; in the majority of the population, the APP-GFP signal at the Golgi was not visible (B.II ), while in a minor percentage of cells APP-GFP was still visible at the Golgi (B.III) . Bar, 10 μm. (C) S655E APP-GFP turnover rate in CHX upon cells pre-incubation with 5nM VPS35 siRNAs for 24 h. Left: Immunoblot analysis of COS-7 transfected cells lysates using the anti-APP 22C11 and anti-tubulin antibodies. Bands a and b, immature and mature APP-GFP forms, respectively, migrating above endogenous APP forms. Right: mature S655E levels were quantified and plotted as percentages of OD values at 0 h in CHX. Values are mean ± SEM (n = 3); (#), statistical significance of p ≤ 0.05 for siRNA VPS35 plus vs control data.

    Journal: Molecular Neurodegeneration

    Article Title: Retrieval of the Alzheimer's amyloid precursor protein from the endosome to the TGN is S655 phosphorylation state-dependent and retromer-mediated

    doi: 10.1186/1750-1326-5-40

    Figure Lengend Snippet: VPS35 down-regulation impairs S655E APP-GFP retrieval to the TGN and decreases its turnover rate . (A) Left: Immunodetection of VPS35 levels in COS-7 cells co-transfected with N1 EGFP and several concentrations of anti-VPS35 siRNAs for 24 h. Tubulin was probed as a control. Right: Quantification of VPS35 levels, plotted as percentages of control conditions (cells only transfected with N1 EGFP). Values are mean ± SEM (n = 2-6); (#), statistical significance of p ≤ 0.05 for siRNA VPS35 vs control data. (B) The 15 min 22C11 antibody uptake assay (Alexa350 blue staining) was applied to monitor S655E APP-GFP protein retrieval from the endosome to the TGN upon COS-7 cells pre-incubation with 5nM VPS35 siRNAs for 24 h to downregulate VPS35 (Texas red staining). (B.I) Control: cells only transfected with S655E APP-GFP. (B.II - B.III) siRNA VPS35: cells co-transfected with S655E APP-GFP and VPS35 siRNAs; in the majority of the population, the APP-GFP signal at the Golgi was not visible (B.II ), while in a minor percentage of cells APP-GFP was still visible at the Golgi (B.III) . Bar, 10 μm. (C) S655E APP-GFP turnover rate in CHX upon cells pre-incubation with 5nM VPS35 siRNAs for 24 h. Left: Immunoblot analysis of COS-7 transfected cells lysates using the anti-APP 22C11 and anti-tubulin antibodies. Bands a and b, immature and mature APP-GFP forms, respectively, migrating above endogenous APP forms. Right: mature S655E levels were quantified and plotted as percentages of OD values at 0 h in CHX. Values are mean ± SEM (n = 3); (#), statistical significance of p ≤ 0.05 for siRNA VPS35 plus vs control data.

    Article Snippet: The resultant fragments were digested with endonucleases (Age I and Nru I) and subcloned into the Age I/Sma I restriction sites of the GFP-encoding mammalian expression vector (pEGFP-N1, Clontech) as N-terminal APP-GFP translational fusions.

    Techniques: Immunodetection, Transfection, Staining, Incubation

    Co-immunoprecipitation of APP, VPS35 and SorLA in COS-7 and HEK293 cells . (A) Endogenous APP and VPS35 were co-immunoprecipitated in COS-7 cells using the anti-APP N-terminus 22C11 and the anti-VPS35 antibodies (αVPS35). Negative controls (C) were performed by immunoprecipitating cells with the same secondary antibodies, sepharose- (IP VPS35 control) and agarose- (IP 22C11) linked, respectively. (B) Transfected Wt APP-GFP and endogenous VPS35 co-immunoprecipitate from COS-7 cells using the indicated antibodies (Ab). (C) Transfected Wt, S655A and S655E APP-GFP were immunoprecipitated in COS-7 cells with the anti-GFP antibody, and the co-immunoprecipitated endogenous VPS35 forms were detected with an anti-VPS35 antibody. Asterisk (*), non-specific IgGs bands (IgGs raised in goat) of the IP samples. Non-transfected cells (C(nt)) submitted to the same IP procedures (incubated with primary and agarose-linked secondary antibodies) were used as control in (B) and (C) . (D) HEK293T cells were cotransfected with SorLA cDNA, APP 695 and eGFP (transfection control). Immunoprecipitation was performed using antibodies raised against the C-terminus of APP (369), the N-terminus of SorLA (αSorLA) or using pre-immune serum as negative controls (C). Immunoprecipitation and co-immunoprecipitation were detected by western blot (WB) using the anti-APP 369 antibody and an anti-SorLA C-terminus antibody (αSorLA C-term). Immunoblot analysis included GFP as an additional negative control.

    Journal: Molecular Neurodegeneration

    Article Title: Retrieval of the Alzheimer's amyloid precursor protein from the endosome to the TGN is S655 phosphorylation state-dependent and retromer-mediated

    doi: 10.1186/1750-1326-5-40

    Figure Lengend Snippet: Co-immunoprecipitation of APP, VPS35 and SorLA in COS-7 and HEK293 cells . (A) Endogenous APP and VPS35 were co-immunoprecipitated in COS-7 cells using the anti-APP N-terminus 22C11 and the anti-VPS35 antibodies (αVPS35). Negative controls (C) were performed by immunoprecipitating cells with the same secondary antibodies, sepharose- (IP VPS35 control) and agarose- (IP 22C11) linked, respectively. (B) Transfected Wt APP-GFP and endogenous VPS35 co-immunoprecipitate from COS-7 cells using the indicated antibodies (Ab). (C) Transfected Wt, S655A and S655E APP-GFP were immunoprecipitated in COS-7 cells with the anti-GFP antibody, and the co-immunoprecipitated endogenous VPS35 forms were detected with an anti-VPS35 antibody. Asterisk (*), non-specific IgGs bands (IgGs raised in goat) of the IP samples. Non-transfected cells (C(nt)) submitted to the same IP procedures (incubated with primary and agarose-linked secondary antibodies) were used as control in (B) and (C) . (D) HEK293T cells were cotransfected with SorLA cDNA, APP 695 and eGFP (transfection control). Immunoprecipitation was performed using antibodies raised against the C-terminus of APP (369), the N-terminus of SorLA (αSorLA) or using pre-immune serum as negative controls (C). Immunoprecipitation and co-immunoprecipitation were detected by western blot (WB) using the anti-APP 369 antibody and an anti-SorLA C-terminus antibody (αSorLA C-term). Immunoblot analysis included GFP as an additional negative control.

    Article Snippet: The resultant fragments were digested with endonucleases (Age I and Nru I) and subcloned into the Age I/Sma I restriction sites of the GFP-encoding mammalian expression vector (pEGFP-N1, Clontech) as N-terminal APP-GFP translational fusions.

    Techniques: Immunoprecipitation, Transfection, Incubation, Western Blot, Negative Control

    A pool of endocytic APP-GFP is targeted to the TGN . Wt APP-GFP at COS-7 cell surface was labelled by incubating cells with the 22C11 anti-APP ectodomain antibody (

    Journal: Molecular Neurodegeneration

    Article Title: Retrieval of the Alzheimer's amyloid precursor protein from the endosome to the TGN is S655 phosphorylation state-dependent and retromer-mediated

    doi: 10.1186/1750-1326-5-40

    Figure Lengend Snippet: A pool of endocytic APP-GFP is targeted to the TGN . Wt APP-GFP at COS-7 cell surface was labelled by incubating cells with the 22C11 anti-APP ectodomain antibody ("22C11 Uptake", Texas red staining) at 4°C. At 0 min, 22C11 staining is maintained at the cell surface (microphotographs taken at the plasma membrane focal plane). Following 15 min at 37°C, part of the 22C11 population can be observed at vesicular structures around the Golgi (arrowhead; microphotographs taken at the Golgi focal plane). Endocytic 22C11/APP-GFP targeting to the TGN was confirmed by co-localization with a pECFP-Golgi construct ("CFP-Golgi", blue fluorescence), a construct targeted to the trans and medial region of the Golgi. Of note, since the CFP-Golgi fusion protein has a weak blue fluorescence, it is virtually non-visible at the plasma membrane focal plane. ROI - region of interest, 2.0-fold magnified. Several white vesicles, denoting 22C11/APP-GFP/CFP-Golgi co-localization, can be observed at the TGN (arrows in ROIs), A tubular morphology can be depicted for some of these vesicles in the ROIs. Bar, 10 μm.

    Article Snippet: The resultant fragments were digested with endonucleases (Age I and Nru I) and subcloned into the Age I/Sma I restriction sites of the GFP-encoding mammalian expression vector (pEGFP-N1, Clontech) as N-terminal APP-GFP translational fusions.

    Techniques: Staining, Construct, Fluorescence

    Endocytosed APP is present in Clathrin and Rab 5-positive vesicles tubulating vesicles . The 22C11 uptake assay (Alexa350 blue staining) was repeated in COS-7 cells for further characterization of the vesicles retrieving endocytosed APP-GFP to the TGN. Immunocytochemistry analysis using antibodies against (A) clathrin and (B) Rab 5 (Texas red staining) confirmed the presence of these proteins in APP-GFP endocytic vesicles. ROI, region of interest, 1.7- and 2.0-fold magnified, respectively. Arrows in ROIs depict APP tubulating endosomes, further magnified. While 22C11/APP-GFP and clathrin were observed to be sorted from the vacuole to the emerging tubule, Rab 5 appears to be maintained in the vacuole. Bar, 10 μm. (C) confocal quantitative analysis of the degree of co-localization between Wt APP-GPP and uptaken 22C11 with clathrin and Rab-5. Values are mean ± SEM, n = 20 cells.

    Journal: Molecular Neurodegeneration

    Article Title: Retrieval of the Alzheimer's amyloid precursor protein from the endosome to the TGN is S655 phosphorylation state-dependent and retromer-mediated

    doi: 10.1186/1750-1326-5-40

    Figure Lengend Snippet: Endocytosed APP is present in Clathrin and Rab 5-positive vesicles tubulating vesicles . The 22C11 uptake assay (Alexa350 blue staining) was repeated in COS-7 cells for further characterization of the vesicles retrieving endocytosed APP-GFP to the TGN. Immunocytochemistry analysis using antibodies against (A) clathrin and (B) Rab 5 (Texas red staining) confirmed the presence of these proteins in APP-GFP endocytic vesicles. ROI, region of interest, 1.7- and 2.0-fold magnified, respectively. Arrows in ROIs depict APP tubulating endosomes, further magnified. While 22C11/APP-GFP and clathrin were observed to be sorted from the vacuole to the emerging tubule, Rab 5 appears to be maintained in the vacuole. Bar, 10 μm. (C) confocal quantitative analysis of the degree of co-localization between Wt APP-GPP and uptaken 22C11 with clathrin and Rab-5. Values are mean ± SEM, n = 20 cells.

    Article Snippet: The resultant fragments were digested with endonucleases (Age I and Nru I) and subcloned into the Age I/Sma I restriction sites of the GFP-encoding mammalian expression vector (pEGFP-N1, Clontech) as N-terminal APP-GFP translational fusions.

    Techniques: Staining, Immunocytochemistry

    S655 APP phosphomutants exhibit differential cellular catabolism . (A) Wt and S655 phosphomutants turnover rates in COS-7 cells. Upper panel: Immunoblot analysis of APP-GFP transfected cells lysates using the anti-APP antibody 22C11. Bands a and b, immature (N-glycosylated) and mature (N- and O-glycosylated) APP-GFP forms, respectively, migrating above endogenous APP forms of ~115 kD and ~109 kD (potentially immature APP 751/770 and APP 695 , respectively). APP-GFP bands identity was further confirmed using an anti-GFP antibody (data not shown). Lower panel: plotted data of the levels of immature (left graph) and mature (right graph) APP-GFP, expressed as percentage of OD values at 0 h in CHX. Values are mean ± SEM (n = 6). Statistical significance symbols: (*), S655 phosphomutants vs. Wt; ( + ), S655A vs. S655E; statistical significance levels are presented as (*/ + ) for p ≤ 0.05; ( ++ ), for p

    Journal: Molecular Neurodegeneration

    Article Title: Retrieval of the Alzheimer's amyloid precursor protein from the endosome to the TGN is S655 phosphorylation state-dependent and retromer-mediated

    doi: 10.1186/1750-1326-5-40

    Figure Lengend Snippet: S655 APP phosphomutants exhibit differential cellular catabolism . (A) Wt and S655 phosphomutants turnover rates in COS-7 cells. Upper panel: Immunoblot analysis of APP-GFP transfected cells lysates using the anti-APP antibody 22C11. Bands a and b, immature (N-glycosylated) and mature (N- and O-glycosylated) APP-GFP forms, respectively, migrating above endogenous APP forms of ~115 kD and ~109 kD (potentially immature APP 751/770 and APP 695 , respectively). APP-GFP bands identity was further confirmed using an anti-GFP antibody (data not shown). Lower panel: plotted data of the levels of immature (left graph) and mature (right graph) APP-GFP, expressed as percentage of OD values at 0 h in CHX. Values are mean ± SEM (n = 6). Statistical significance symbols: (*), S655 phosphomutants vs. Wt; ( + ), S655A vs. S655E; statistical significance levels are presented as (*/ + ) for p ≤ 0.05; ( ++ ), for p

    Article Snippet: The resultant fragments were digested with endonucleases (Age I and Nru I) and subcloned into the Age I/Sma I restriction sites of the GFP-encoding mammalian expression vector (pEGFP-N1, Clontech) as N-terminal APP-GFP translational fusions.

    Techniques: Transfection

    S655 phosphorylation-dependent APP retrieval occurs via VPS35-containing endocytic vesicles . (A) APP-GFP transfected COS-7 cells were subjected to the 15 min 22C11 uptake assay (Alexa350 blue staining) and the co-localization of endocytosed APP-GFP proteins (blue/green vesicles) with the VPS35 retromer subunit (Texas red staining) was monitored. The Golgi area denotes the highest degree of co-localization, and a region was magnified for better visualization of the tubulating vesicles (ROI-4.0-fold magnified; arrows point to APP-GFP/AU22C11/VPS35-positive structures for Wt and S655E, and arrowhead to a S655A APP-GFP-negative, AU22C11/VPS35-positive structure). Some of the tubulating vesicles for Wt and S655E have been represented schematically with a black line. (B) Co-localization between APP-GFP or 22C11 uptake with VPS35 was quantified using confocal software. Values are mean ± SEM, n = 30 cells. Statistical significance symbols: (*), S655 phosphomutant vs Wt data; ( + ), S655A vs S655E data. Statistically significant levels are presented as (**) for p

    Journal: Molecular Neurodegeneration

    Article Title: Retrieval of the Alzheimer's amyloid precursor protein from the endosome to the TGN is S655 phosphorylation state-dependent and retromer-mediated

    doi: 10.1186/1750-1326-5-40

    Figure Lengend Snippet: S655 phosphorylation-dependent APP retrieval occurs via VPS35-containing endocytic vesicles . (A) APP-GFP transfected COS-7 cells were subjected to the 15 min 22C11 uptake assay (Alexa350 blue staining) and the co-localization of endocytosed APP-GFP proteins (blue/green vesicles) with the VPS35 retromer subunit (Texas red staining) was monitored. The Golgi area denotes the highest degree of co-localization, and a region was magnified for better visualization of the tubulating vesicles (ROI-4.0-fold magnified; arrows point to APP-GFP/AU22C11/VPS35-positive structures for Wt and S655E, and arrowhead to a S655A APP-GFP-negative, AU22C11/VPS35-positive structure). Some of the tubulating vesicles for Wt and S655E have been represented schematically with a black line. (B) Co-localization between APP-GFP or 22C11 uptake with VPS35 was quantified using confocal software. Values are mean ± SEM, n = 30 cells. Statistical significance symbols: (*), S655 phosphomutant vs Wt data; ( + ), S655A vs S655E data. Statistically significant levels are presented as (**) for p

    Article Snippet: The resultant fragments were digested with endonucleases (Age I and Nru I) and subcloned into the Age I/Sma I restriction sites of the GFP-encoding mammalian expression vector (pEGFP-N1, Clontech) as N-terminal APP-GFP translational fusions.

    Techniques: Transfection, Staining, Software

    APP-GFP traverses the endocytic pathway . (a) COS-7 cells transfected with Wt APP-GFP were exposed for 3 h to the protein synthesis inhibitor CHX and incubated in the last 15 min with Texas Red-conjugated transferring. Co-localization (yellow/orange fluorescence vesicles) between transferrin (Texas red endocytic vesicles) and APP-GFP green cytoplasmic vesicles can be observed (Overlay panel). Immunocytochemistry analysis with (b) an early (Rab 5) and (c) late (Rab 7) endosomal markers were also performed and co-localization observed. ROI, region of interest, denotes APP-GFP co-localizing endocytic vesicles. Bar, 10 μm. Fluorescence intensity profiles are also presented, representing the voxels through the white arrowed lines indicated in the ROI overlay images; asterisks denote co-localizing vesicles. A microphotograph of the eGFP protein is presented as a negative control, GFP alone is distributed through the nucleus and cytoplasm, but is not targeted to cytoplasmic vesicles.

    Journal: Molecular Neurodegeneration

    Article Title: Retrieval of the Alzheimer's amyloid precursor protein from the endosome to the TGN is S655 phosphorylation state-dependent and retromer-mediated

    doi: 10.1186/1750-1326-5-40

    Figure Lengend Snippet: APP-GFP traverses the endocytic pathway . (a) COS-7 cells transfected with Wt APP-GFP were exposed for 3 h to the protein synthesis inhibitor CHX and incubated in the last 15 min with Texas Red-conjugated transferring. Co-localization (yellow/orange fluorescence vesicles) between transferrin (Texas red endocytic vesicles) and APP-GFP green cytoplasmic vesicles can be observed (Overlay panel). Immunocytochemistry analysis with (b) an early (Rab 5) and (c) late (Rab 7) endosomal markers were also performed and co-localization observed. ROI, region of interest, denotes APP-GFP co-localizing endocytic vesicles. Bar, 10 μm. Fluorescence intensity profiles are also presented, representing the voxels through the white arrowed lines indicated in the ROI overlay images; asterisks denote co-localizing vesicles. A microphotograph of the eGFP protein is presented as a negative control, GFP alone is distributed through the nucleus and cytoplasm, but is not targeted to cytoplasmic vesicles.

    Article Snippet: The resultant fragments were digested with endonucleases (Age I and Nru I) and subcloned into the Age I/Sma I restriction sites of the GFP-encoding mammalian expression vector (pEGFP-N1, Clontech) as N-terminal APP-GFP translational fusions.

    Techniques: Transfection, Incubation, Transferring, Fluorescence, Immunocytochemistry, Negative Control

    Apoptosis assay with CHO cells expressing scFv-caspase and specific antigen. ( A ) CHO cells were cotransfected with pEGFP-N1 and clones encoding various scFv fusions. Transfected cells were identified by fluorescence and sorted with a FACSCalibur cell sorter. Genomic DNA was extracted from selected cells and PCR amplifications carried out with the ApoAlert LM-PCR Ladder Assay Kit. The products were fractionated on 1.5% agarose. The lanes correspond to PCRs from DNA of cells that had been transfected with plasmids expressing scFvR4-VP16 + β-gal (lane 1), scFvR4-caspase 3 + β-gal (lane 2), scFvR4-caspase 3(C163S) + β-gal (lane 3), scFvF8-caspase 3 + β-gal (lane 4), or scFvR4-caspase 3 alone (lane 5). The negative PCR control represents a product from a reaction identical with the others but lacking template DNA. ( B ) PCRs were performed with the same DNA samples as in A , but using primers specific for the Chinese hamster actin gene. The negative control was as in A ; the positive control was the reaction product obtained from a PCR amplification with purified CHO genomic DNA.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Intracellular antibody-caspase-mediated cell killing: An approach for application in cancer therapy

    doi:

    Figure Lengend Snippet: Apoptosis assay with CHO cells expressing scFv-caspase and specific antigen. ( A ) CHO cells were cotransfected with pEGFP-N1 and clones encoding various scFv fusions. Transfected cells were identified by fluorescence and sorted with a FACSCalibur cell sorter. Genomic DNA was extracted from selected cells and PCR amplifications carried out with the ApoAlert LM-PCR Ladder Assay Kit. The products were fractionated on 1.5% agarose. The lanes correspond to PCRs from DNA of cells that had been transfected with plasmids expressing scFvR4-VP16 + β-gal (lane 1), scFvR4-caspase 3 + β-gal (lane 2), scFvR4-caspase 3(C163S) + β-gal (lane 3), scFvF8-caspase 3 + β-gal (lane 4), or scFvR4-caspase 3 alone (lane 5). The negative PCR control represents a product from a reaction identical with the others but lacking template DNA. ( B ) PCRs were performed with the same DNA samples as in A , but using primers specific for the Chinese hamster actin gene. The negative control was as in A ; the positive control was the reaction product obtained from a PCR amplification with purified CHO genomic DNA.

    Article Snippet: pM-βgal, pNL-scFvR4-VP16, pNL-scFvF8-VP16, and pNL-scFv-IN33-VP16 were described previously ( ). pRSV-Luc (firefly luciferase expression vector) was also described previously ( ) and pEGFP-N1 [enhanced green fluorescence protein (GFP) expression vector] was commercially available (CLONTECH).

    Techniques: Apoptosis Assay, Expressing, Clone Assay, Transfection, Fluorescence, Polymerase Chain Reaction, Negative Control, Positive Control, Amplification, Purification

    Decreased dendritic complexity in DSCAM-overexpressing hippocampal neurons. A , Representative micrographs of hippocampal neurons transfected at DIV7 with the indicated constructs. Neuronal morphology was visualized by GFP immunocytochemistry 2 d after transfection. Dendrites were identified as MAP2-positive neurites. Scale bars, 60 μm. B , Sholl analysis of the neurons transfected with GFP or DSCAM-IRES-GFP vectors. C , Total dendritic length of neurons transfected with the indicated constructs. D , Number of primary dendrites of neurons transfected with the indicated constructs. E , Somatic area of neurons transfected with the indicated plasmids. Mean values are shown (12–15 neurons); the error bars indicate the SEM, and asterisks denote statistically significant differences: * p

    Journal: The Journal of Neuroscience

    Article Title: NMDA-Mediated Regulation of DSCAM Dendritic Local Translation Is Lost in a Mouse Model of Down's Syndrome

    doi: 10.1523/JNEUROSCI.3457-10.2010

    Figure Lengend Snippet: Decreased dendritic complexity in DSCAM-overexpressing hippocampal neurons. A , Representative micrographs of hippocampal neurons transfected at DIV7 with the indicated constructs. Neuronal morphology was visualized by GFP immunocytochemistry 2 d after transfection. Dendrites were identified as MAP2-positive neurites. Scale bars, 60 μm. B , Sholl analysis of the neurons transfected with GFP or DSCAM-IRES-GFP vectors. C , Total dendritic length of neurons transfected with the indicated constructs. D , Number of primary dendrites of neurons transfected with the indicated constructs. E , Somatic area of neurons transfected with the indicated plasmids. Mean values are shown (12–15 neurons); the error bars indicate the SEM, and asterisks denote statistically significant differences: * p

    Article Snippet: A total of 0.8 μg of a plasmid expressing mouse DSCAM along with green fluorescent protein (GFP) protein (CMV-DSCAM-IRES-GFP) ( ) or a control vector expressing GFP protein (pEGFP-C1; Clontech) was transfected per well.

    Techniques: Transfection, Construct, Immunocytochemistry