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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 (
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1) Product Images from "Retrieval of the Alzheimer's amyloid precursor protein from the endosome to the TGN is S655 phosphorylation state-dependent and retromer-mediated"

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

Journal: Molecular Neurodegeneration

doi: 10.1186/1750-1326-5-40

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 (
Figure Legend 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

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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 (
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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.
Figure Legend 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.

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

Related Articles

Expressing:

Article Title: Retrieval of the Alzheimer's amyloid precursor protein from the endosome to the TGN is S655 phosphorylation state-dependent and retromer-mediated
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. .. The nucleotide sequences of the APP695 phosphorylation cDNA point mutants and the open reading frames were confirmed by DNA sequencing (ABI PRISM 310 genetic Analyser, Applied Biosystems).

Plasmid Preparation:

Article Title: Retrieval of the Alzheimer's amyloid precursor protein from the endosome to the TGN is S655 phosphorylation state-dependent and retromer-mediated
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. .. The nucleotide sequences of the APP695 phosphorylation cDNA point mutants and the open reading frames were confirmed by DNA sequencing (ABI PRISM 310 genetic Analyser, Applied Biosystems).

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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
    https://www.bioz.com/result/gfp encoding mammalian expression vector/product/TaKaRa
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    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