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mouse monoclonal anti vapa 4c12  (Santa Cruz Biotechnology)


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    Santa Cruz Biotechnology mouse monoclonal anti vapa 4c12
    Mouse Monoclonal Anti Vapa 4c12, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mouse+anti+vapa/VAP-A+Antibody/bio_rxiv__2025__10__23__684214-226-98-102
    Average 93 stars, based on 41 article reviews
    mouse monoclonal anti vapa 4c12 - by Bioz Stars, 2026-10
    93/100 stars

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    Incubation:

    Article Title: Homotypic SCOTIN assemblies form ER ‐endosome membrane contacts and regulate endosome dynamics
    Article Snippet: Proximity ligation assay was performed in a humid chamber with a kit according to the manufacturer’s instructions (Sigma–Aldrich, DUO92101). .. The samples were incubated with mouse anti-VAPA (Santa Cruz, sc-293278), mouse RTN4 (Santa Cruz, sc-271878), or rabbit anti-Rab7 (Cell Signaling Technology, 9367S) overnight at 4°C, and the negative control samples were incubated with single antibodies alone. .. After washing, the cells were incubated with antirabbit PLA PLUS and anti-mouse PLA MINUS probes (Duolink; Sigma–Aldrich, DUO92002 and DUO92004) for 1 h at 37°C in a humidified chamber.

    Article Title: Homotypic SCOTIN assemblies form ER-endosome membrane contacts and regulate endosome dynamics.
    Article Snippet: Proximity ligation assay was performed in a humid chamber with a kit according to the manufacturer’s instructions (Sigma–Aldrich, DUO92101). .. The samples were incubated with mouse anti-VAPA (Santa Cruz, sc-293278), mouse RTN4 (Santa Cruz, sc-271878), or rabbit anti-Rab7 (Cell Signaling Technology, 9367S) overnight at 4°C, and the negative control samples were incubated with single antibodies alone. .. After washing, the cells were incubated with antirabbit PLA PLUS and anti-mouse PLA MINUS probes (Duolink; Sigma–Aldrich, DUO92002 and DUO92004) for 1 h at 37°C in a humidified chamber.

    Negative Control:

    Article Title: Homotypic SCOTIN assemblies form ER ‐endosome membrane contacts and regulate endosome dynamics
    Article Snippet: Proximity ligation assay was performed in a humid chamber with a kit according to the manufacturer’s instructions (Sigma–Aldrich, DUO92101). .. The samples were incubated with mouse anti-VAPA (Santa Cruz, sc-293278), mouse RTN4 (Santa Cruz, sc-271878), or rabbit anti-Rab7 (Cell Signaling Technology, 9367S) overnight at 4°C, and the negative control samples were incubated with single antibodies alone. .. After washing, the cells were incubated with antirabbit PLA PLUS and anti-mouse PLA MINUS probes (Duolink; Sigma–Aldrich, DUO92002 and DUO92004) for 1 h at 37°C in a humidified chamber.

    Article Title: Homotypic SCOTIN assemblies form ER-endosome membrane contacts and regulate endosome dynamics.
    Article Snippet: Proximity ligation assay was performed in a humid chamber with a kit according to the manufacturer’s instructions (Sigma–Aldrich, DUO92101). .. The samples were incubated with mouse anti-VAPA (Santa Cruz, sc-293278), mouse RTN4 (Santa Cruz, sc-271878), or rabbit anti-Rab7 (Cell Signaling Technology, 9367S) overnight at 4°C, and the negative control samples were incubated with single antibodies alone. .. After washing, the cells were incubated with antirabbit PLA PLUS and anti-mouse PLA MINUS probes (Duolink; Sigma–Aldrich, DUO92002 and DUO92004) for 1 h at 37°C in a humidified chamber.



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    mouse monoclonal anti vapa 4c12  (Santa Cruz Biotechnology)
    93
    Santa Cruz Biotechnology mouse monoclonal anti vapa 4c12
    Mouse Monoclonal Anti Vapa 4c12, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mouse+anti+vapa/VAP-A+Antibody/bio_rxiv__2025__10__23__684214-226-98-102
    Average 93 stars, based on 1 article reviews
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    mouse anti-vapa monoclonal antibody mabn361  (Millipore)
    90
    Millipore mouse anti-vapa monoclonal antibody mabn361
    BMM were infected with L. amazonensis or L. major metacyclic promastigotes for the indicated time points. Controls consisted of untreated BMM and BMM fed with zymosan. (A) Distribution of <t>VAPA</t> (green) and its association with PVs were assessed by confocal immunofluorescence microscopy. DNA is shown in blue. (B) Quantification of the association of VAPA to L. amazonensis- and L. major -harboring PVs and to zymosan-containing phagosomes. (C) Colocalization of LAMP1 with VAPA on communal PVs harboring L.amazonensis at 48 h post-infection. Colocalization (white pixels) of LAMP-1 (red) with VAPA (green) was assessed and quantified by confocal immunofluorescence microscopy. Insets display the PV area. DNA is shown in blue. (D) Western blot analysis of VAPA levels over time in L. amazonensis -infected BMM. Data are presented as the means ± standard errors of the means (SEM) of values from three independent experiments. White arrowheads denote internalized parasites. Representative images from three experiments are shown. **, P ≤ 0.01; ***, P ≤ 0.001.
    Mouse Anti Vapa Monoclonal Antibody Mabn361, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mouse+anti+vapa/mouse+anti+vapa+monoclonal+antibody+mabn361/pmc11981147-176-1-8
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    mouse anti vapa monoclonal antibody  (Atlas Antibodies)
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    Atlas Antibodies mouse anti vapa monoclonal antibody
    ( A ) Left panel: Representative immunoblots showing the levels of <t>VAPA</t> and Tubulin in Control and VAPA KO cells. Tubulin expression level was used as loading control. Right panel: quantification of relative VAPA density in Control and VAPA KO cells normalized to Tubulin levels (mean ± SEM from three independent experiments). Data were analysed using a single-sample Student t-test. ( B ) Confocal images of Control and VAPA KO cells immunostained for VAPA. Scale bar 20 µm. ( C ) Confocal images of VAPA KO leader cells expressing wild-type mCherry-VAPA (VAPA KO +WT) or mCherry-VAPA KD/MD (VAPA KO +KDMD). Scale bar 10 µm. ( D ) Phase contrast images of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells migrating collectively 48 hr after space release. For each condition, the trajectory of a leader cell over 20 hr is shown in green. Scale bar: 100 µm. ( E ) Analysis of relative colonized surface within the last 20 h by Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells (mean ± SEM from three independent experiments). Data were analysed using a One-way Anova paired test. ( F, G ) Analysis of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cell velocity ( F ) n=40, 40, 3,9 and 39 cells respectively from three independent experiments and directionality coefficient ( G ) n=34, 36, 32, and 26 cells respectively from three independent experiments. Data were analysed using a One-way Anova Kruskal-Wallis test. ( H ) Phase contrast images of a Control and VAPA KO individual cell displacing on fibronectin-coated glass during 5 minutes. The cell trajectory is shown in blue. Scale bar: 20 µm. ( I, J ) Analysis of cell velocity ( I ) and directionality coefficient ( J ) of Control and VAPA KO individual cells displacing on fibronectin-coated glass during at least 3 h (mean ± SEM; Control: n=64 cells; VAPA KO: n=33 cells from two independent experiments). data were analysed using non parametric Mann-Whitney t-test. ( K ) Epifluorescence images of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells after 1 hour spreading on fibronectin-coated glass and stained as indicated. Scale bar: 50 µm. ( L ) Analysis of cells area after 1 hour spreading on fibronectin-coated glass (mean ± SEM; n=117, 134, 113, and 128 cells respectively from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test. (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 1—source data 1. Table containing the raw data used for the quantifications in . Figure 1—source data 2. Folder containing the 2 original files of the full raw unedited blots for VAPA and Tubulin presented in figure with the uncropped annotated blots.
    Mouse Anti Vapa Monoclonal Antibody, supplied by Atlas Antibodies, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse igg anti vapa  (Santa Cruz Biotechnology)
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    Santa Cruz Biotechnology mouse igg anti vapa
    ( A ) Left panel: Representative immunoblots showing the levels of <t>VAPA</t> and Tubulin in Control and VAPA KO cells. Tubulin expression level was used as loading control. Right panel: quantification of relative VAPA density in Control and VAPA KO cells normalized to Tubulin levels (mean ± SEM from three independent experiments). Data were analysed using a single-sample Student t-test. ( B ) Confocal images of Control and VAPA KO cells immunostained for VAPA. Scale bar 20 µm. ( C ) Confocal images of VAPA KO leader cells expressing wild-type mCherry-VAPA (VAPA KO +WT) or mCherry-VAPA KD/MD (VAPA KO +KDMD). Scale bar 10 µm. ( D ) Phase contrast images of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells migrating collectively 48 hr after space release. For each condition, the trajectory of a leader cell over 20 hr is shown in green. Scale bar: 100 µm. ( E ) Analysis of relative colonized surface within the last 20 h by Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells (mean ± SEM from three independent experiments). Data were analysed using a One-way Anova paired test. ( F, G ) Analysis of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cell velocity ( F ) n=40, 40, 3,9 and 39 cells respectively from three independent experiments and directionality coefficient ( G ) n=34, 36, 32, and 26 cells respectively from three independent experiments. Data were analysed using a One-way Anova Kruskal-Wallis test. ( H ) Phase contrast images of a Control and VAPA KO individual cell displacing on fibronectin-coated glass during 5 minutes. The cell trajectory is shown in blue. Scale bar: 20 µm. ( I, J ) Analysis of cell velocity ( I ) and directionality coefficient ( J ) of Control and VAPA KO individual cells displacing on fibronectin-coated glass during at least 3 h (mean ± SEM; Control: n=64 cells; VAPA KO: n=33 cells from two independent experiments). data were analysed using non parametric Mann-Whitney t-test. ( K ) Epifluorescence images of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells after 1 hour spreading on fibronectin-coated glass and stained as indicated. Scale bar: 50 µm. ( L ) Analysis of cells area after 1 hour spreading on fibronectin-coated glass (mean ± SEM; n=117, 134, 113, and 128 cells respectively from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test. (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 1—source data 1. Table containing the raw data used for the quantifications in . Figure 1—source data 2. Folder containing the 2 original files of the full raw unedited blots for VAPA and Tubulin presented in figure with the uncropped annotated blots.
    Mouse Igg Anti Vapa, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse anti vapa  (Santa Cruz Biotechnology)
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    Santa Cruz Biotechnology mouse anti vapa
    ( A ) Left panel: Representative immunoblots showing the levels of <t>VAPA</t> and Tubulin in Control and VAPA KO cells. Tubulin expression level was used as loading control. Right panel: quantification of relative VAPA density in Control and VAPA KO cells normalized to Tubulin levels (mean ± SEM from three independent experiments). Data were analysed using a single-sample Student t-test. ( B ) Confocal images of Control and VAPA KO cells immunostained for VAPA. Scale bar 20 µm. ( C ) Confocal images of VAPA KO leader cells expressing wild-type mCherry-VAPA (VAPA KO +WT) or mCherry-VAPA KD/MD (VAPA KO +KDMD). Scale bar 10 µm. ( D ) Phase contrast images of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells migrating collectively 48 hr after space release. For each condition, the trajectory of a leader cell over 20 hr is shown in green. Scale bar: 100 µm. ( E ) Analysis of relative colonized surface within the last 20 h by Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells (mean ± SEM from three independent experiments). Data were analysed using a One-way Anova paired test. ( F, G ) Analysis of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cell velocity ( F ) n=40, 40, 3,9 and 39 cells respectively from three independent experiments and directionality coefficient ( G ) n=34, 36, 32, and 26 cells respectively from three independent experiments. Data were analysed using a One-way Anova Kruskal-Wallis test. ( H ) Phase contrast images of a Control and VAPA KO individual cell displacing on fibronectin-coated glass during 5 minutes. The cell trajectory is shown in blue. Scale bar: 20 µm. ( I, J ) Analysis of cell velocity ( I ) and directionality coefficient ( J ) of Control and VAPA KO individual cells displacing on fibronectin-coated glass during at least 3 h (mean ± SEM; Control: n=64 cells; VAPA KO: n=33 cells from two independent experiments). data were analysed using non parametric Mann-Whitney t-test. ( K ) Epifluorescence images of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells after 1 hour spreading on fibronectin-coated glass and stained as indicated. Scale bar: 50 µm. ( L ) Analysis of cells area after 1 hour spreading on fibronectin-coated glass (mean ± SEM; n=117, 134, 113, and 128 cells respectively from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test. (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 1—source data 1. Table containing the raw data used for the quantifications in . Figure 1—source data 2. Folder containing the 2 original files of the full raw unedited blots for VAPA and Tubulin presented in figure with the uncropped annotated blots.
    Mouse Anti Vapa, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mouse+anti+vapa/VAP-A+Antibody/pm37377038-489-5-7
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    mouse anti-vapa monoclonal antibody  (NeuroMab)
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    NeuroMab mouse anti-vapa monoclonal antibody
    ( A ) Left panel: Representative immunoblots showing the levels of <t>VAPA</t> and Tubulin in Control and VAPA KO cells. Tubulin expression level was used as loading control. Right panel: quantification of relative VAPA density in Control and VAPA KO cells normalized to Tubulin levels (mean ± SEM from three independent experiments). Data were analysed using a single-sample Student t-test. ( B ) Confocal images of Control and VAPA KO cells immunostained for VAPA. Scale bar 20 µm. ( C ) Confocal images of VAPA KO leader cells expressing wild-type mCherry-VAPA (VAPA KO +WT) or mCherry-VAPA KD/MD (VAPA KO +KDMD). Scale bar 10 µm. ( D ) Phase contrast images of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells migrating collectively 48 hr after space release. For each condition, the trajectory of a leader cell over 20 hr is shown in green. Scale bar: 100 µm. ( E ) Analysis of relative colonized surface within the last 20 h by Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells (mean ± SEM from three independent experiments). Data were analysed using a One-way Anova paired test. ( F, G ) Analysis of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cell velocity ( F ) n=40, 40, 3,9 and 39 cells respectively from three independent experiments and directionality coefficient ( G ) n=34, 36, 32, and 26 cells respectively from three independent experiments. Data were analysed using a One-way Anova Kruskal-Wallis test. ( H ) Phase contrast images of a Control and VAPA KO individual cell displacing on fibronectin-coated glass during 5 minutes. The cell trajectory is shown in blue. Scale bar: 20 µm. ( I, J ) Analysis of cell velocity ( I ) and directionality coefficient ( J ) of Control and VAPA KO individual cells displacing on fibronectin-coated glass during at least 3 h (mean ± SEM; Control: n=64 cells; VAPA KO: n=33 cells from two independent experiments). data were analysed using non parametric Mann-Whitney t-test. ( K ) Epifluorescence images of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells after 1 hour spreading on fibronectin-coated glass and stained as indicated. Scale bar: 50 µm. ( L ) Analysis of cells area after 1 hour spreading on fibronectin-coated glass (mean ± SEM; n=117, 134, 113, and 128 cells respectively from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test. (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 1—source data 1. Table containing the raw data used for the quantifications in . Figure 1—source data 2. Folder containing the 2 original files of the full raw unedited blots for VAPA and Tubulin presented in figure with the uncropped annotated blots.
    Mouse Anti Vapa Monoclonal Antibody, supplied by NeuroMab, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti vapa mouse monoclonal  (Santa Cruz Biotechnology)
    93
    Santa Cruz Biotechnology anti vapa mouse monoclonal
    ( A ) Left panel: Representative immunoblots showing the levels of <t>VAPA</t> and Tubulin in Control and VAPA KO cells. Tubulin expression level was used as loading control. Right panel: quantification of relative VAPA density in Control and VAPA KO cells normalized to Tubulin levels (mean ± SEM from three independent experiments). Data were analysed using a single-sample Student t-test. ( B ) Confocal images of Control and VAPA KO cells immunostained for VAPA. Scale bar 20 µm. ( C ) Confocal images of VAPA KO leader cells expressing wild-type mCherry-VAPA (VAPA KO +WT) or mCherry-VAPA KD/MD (VAPA KO +KDMD). Scale bar 10 µm. ( D ) Phase contrast images of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells migrating collectively 48 hr after space release. For each condition, the trajectory of a leader cell over 20 hr is shown in green. Scale bar: 100 µm. ( E ) Analysis of relative colonized surface within the last 20 h by Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells (mean ± SEM from three independent experiments). Data were analysed using a One-way Anova paired test. ( F, G ) Analysis of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cell velocity ( F ) n=40, 40, 3,9 and 39 cells respectively from three independent experiments and directionality coefficient ( G ) n=34, 36, 32, and 26 cells respectively from three independent experiments. Data were analysed using a One-way Anova Kruskal-Wallis test. ( H ) Phase contrast images of a Control and VAPA KO individual cell displacing on fibronectin-coated glass during 5 minutes. The cell trajectory is shown in blue. Scale bar: 20 µm. ( I, J ) Analysis of cell velocity ( I ) and directionality coefficient ( J ) of Control and VAPA KO individual cells displacing on fibronectin-coated glass during at least 3 h (mean ± SEM; Control: n=64 cells; VAPA KO: n=33 cells from two independent experiments). data were analysed using non parametric Mann-Whitney t-test. ( K ) Epifluorescence images of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells after 1 hour spreading on fibronectin-coated glass and stained as indicated. Scale bar: 50 µm. ( L ) Analysis of cells area after 1 hour spreading on fibronectin-coated glass (mean ± SEM; n=117, 134, 113, and 128 cells respectively from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test. (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 1—source data 1. Table containing the raw data used for the quantifications in . Figure 1—source data 2. Folder containing the 2 original files of the full raw unedited blots for VAPA and Tubulin presented in figure with the uncropped annotated blots.
    Anti Vapa Mouse Monoclonal, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mouse+anti+vapa/VAP-A+Antibody/pm36627747-274-22-28
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    Image Search Results


    BMM were infected with L. amazonensis or L. major metacyclic promastigotes for the indicated time points. Controls consisted of untreated BMM and BMM fed with zymosan. (A) Distribution of VAPA (green) and its association with PVs were assessed by confocal immunofluorescence microscopy. DNA is shown in blue. (B) Quantification of the association of VAPA to L. amazonensis- and L. major -harboring PVs and to zymosan-containing phagosomes. (C) Colocalization of LAMP1 with VAPA on communal PVs harboring L.amazonensis at 48 h post-infection. Colocalization (white pixels) of LAMP-1 (red) with VAPA (green) was assessed and quantified by confocal immunofluorescence microscopy. Insets display the PV area. DNA is shown in blue. (D) Western blot analysis of VAPA levels over time in L. amazonensis -infected BMM. Data are presented as the means ± standard errors of the means (SEM) of values from three independent experiments. White arrowheads denote internalized parasites. Representative images from three experiments are shown. **, P ≤ 0.01; ***, P ≤ 0.001.

    Journal: PLOS Pathogens

    Article Title: VAPA mediates lipid exchange between Leishmania amazonensis and host macrophages

    doi: 10.1371/journal.ppat.1012636

    Figure Lengend Snippet: BMM were infected with L. amazonensis or L. major metacyclic promastigotes for the indicated time points. Controls consisted of untreated BMM and BMM fed with zymosan. (A) Distribution of VAPA (green) and its association with PVs were assessed by confocal immunofluorescence microscopy. DNA is shown in blue. (B) Quantification of the association of VAPA to L. amazonensis- and L. major -harboring PVs and to zymosan-containing phagosomes. (C) Colocalization of LAMP1 with VAPA on communal PVs harboring L.amazonensis at 48 h post-infection. Colocalization (white pixels) of LAMP-1 (red) with VAPA (green) was assessed and quantified by confocal immunofluorescence microscopy. Insets display the PV area. DNA is shown in blue. (D) Western blot analysis of VAPA levels over time in L. amazonensis -infected BMM. Data are presented as the means ± standard errors of the means (SEM) of values from three independent experiments. White arrowheads denote internalized parasites. Representative images from three experiments are shown. **, P ≤ 0.01; ***, P ≤ 0.001.

    Article Snippet: The mouse anti-VAPA monoclonal antibody (MABN361) was from Millipore, the rabbit anti-CERT polyclonal antibody (PA5-115035) was from Invitrogen, the rabbit anti-ORP1L polyclonal antibody ORP1L (ab131165), the rat anti-nSMase2 (ab85017) and the rat anti-BrdU (ab6326) were from Abcam, the rabbit anti-GLTP (10850-1-AP) was from ThermoFisher, the mouse anti-phosphoglycan (Galβ1,4Manα1-PO4) CA7AE monoclonal antibody [ ] was from Cedarlane, the rabbit anti-β-actin polyclonal antibody was from Cell Signalling, and the rat LAMP-1 monoclonal antibody 1D4B developed by J.T.August and purchased through the Developmental Studies Hybridoma Bank at the University of lowa and the National Institute of Child Health and Human Development.

    Techniques: Infection, Immunofluorescence, Microscopy, Western Blot

    BMM (untreated, treated with siRNA to VAPA, or treated with scrambled siRNA) were infected with L. amazonensis or L. major metacyclic promastigotes and at various time points post-phagocytosis parasite replication and PV size were assessed. (A) Quantification of L. amazonensis parasite burden at 2, 24, 48, and 72 h post-infection. Data are presented as the means ± SEM of values from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001. (B) Quantification of L. major parasite burden at 2, 24, 48, and 72 h post-infection. Data are presented as the means ± SEM of values from three independent experiments. *, P ≤ 0.05; **, P ≤ 0.01. (C) Labeling of L. amazonensis amastigotes with BrdU in BMM (untreated or treated with siRNA to VAPA) infected for 48 h and 72 h. (D) Percent of BrdU+ parasites in BMM (untreated, treated with siRNA to VAPA or with scrambled siRNA) at 48 h and 72 h post-phagocytosis. Data are presented as the means ± SEM of values from three independent experiments. ***, P ≤ 0.001. (E) Quantification of PV size in BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. Data are presented as a cloud with means ± standard deviations (SD) of values from three independent experiments for a total of 450 PVs. **, P ≤ 0.01; ***, P ≤ 0.001. Blots showing the efficacy the siRNA-mediated VAPA knockdowns are shown in .

    Journal: PLOS Pathogens

    Article Title: VAPA mediates lipid exchange between Leishmania amazonensis and host macrophages

    doi: 10.1371/journal.ppat.1012636

    Figure Lengend Snippet: BMM (untreated, treated with siRNA to VAPA, or treated with scrambled siRNA) were infected with L. amazonensis or L. major metacyclic promastigotes and at various time points post-phagocytosis parasite replication and PV size were assessed. (A) Quantification of L. amazonensis parasite burden at 2, 24, 48, and 72 h post-infection. Data are presented as the means ± SEM of values from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001. (B) Quantification of L. major parasite burden at 2, 24, 48, and 72 h post-infection. Data are presented as the means ± SEM of values from three independent experiments. *, P ≤ 0.05; **, P ≤ 0.01. (C) Labeling of L. amazonensis amastigotes with BrdU in BMM (untreated or treated with siRNA to VAPA) infected for 48 h and 72 h. (D) Percent of BrdU+ parasites in BMM (untreated, treated with siRNA to VAPA or with scrambled siRNA) at 48 h and 72 h post-phagocytosis. Data are presented as the means ± SEM of values from three independent experiments. ***, P ≤ 0.001. (E) Quantification of PV size in BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. Data are presented as a cloud with means ± standard deviations (SD) of values from three independent experiments for a total of 450 PVs. **, P ≤ 0.01; ***, P ≤ 0.001. Blots showing the efficacy the siRNA-mediated VAPA knockdowns are shown in .

    Article Snippet: The mouse anti-VAPA monoclonal antibody (MABN361) was from Millipore, the rabbit anti-CERT polyclonal antibody (PA5-115035) was from Invitrogen, the rabbit anti-ORP1L polyclonal antibody ORP1L (ab131165), the rat anti-nSMase2 (ab85017) and the rat anti-BrdU (ab6326) were from Abcam, the rabbit anti-GLTP (10850-1-AP) was from ThermoFisher, the mouse anti-phosphoglycan (Galβ1,4Manα1-PO4) CA7AE monoclonal antibody [ ] was from Cedarlane, the rabbit anti-β-actin polyclonal antibody was from Cell Signalling, and the rat LAMP-1 monoclonal antibody 1D4B developed by J.T.August and purchased through the Developmental Studies Hybridoma Bank at the University of lowa and the National Institute of Child Health and Human Development.

    Techniques: Infection, Labeling

    BMM (untreated, treated with siRNA to VAPA, treated with scrambled siRNA, and mock transfected) were infected with L. amazonensis metacyclic promastigotes and at 48 h post-infection BMM were incubated with BODIPY FL-C5-Ceramide (green). (A) Images of live cells were acquired at 5 min and 120 min after the addition of BODIPY FL C5-Ceramide. (B) Quantification of BODIPYFL-C5 positive L. amazonensis amastigotes at 5 min and 120 min after the addition of BODIPY FL-C5-Ceramide. Data are presented with means ± standard deviations (SD) of values from three independent experiments. ***, P ≤ 0.001 (in comparison to controls, siScr and mock). Blots showing the efficacy the siRNA-mediated VAPA knockdowns are shown in .

    Journal: PLOS Pathogens

    Article Title: VAPA mediates lipid exchange between Leishmania amazonensis and host macrophages

    doi: 10.1371/journal.ppat.1012636

    Figure Lengend Snippet: BMM (untreated, treated with siRNA to VAPA, treated with scrambled siRNA, and mock transfected) were infected with L. amazonensis metacyclic promastigotes and at 48 h post-infection BMM were incubated with BODIPY FL-C5-Ceramide (green). (A) Images of live cells were acquired at 5 min and 120 min after the addition of BODIPY FL C5-Ceramide. (B) Quantification of BODIPYFL-C5 positive L. amazonensis amastigotes at 5 min and 120 min after the addition of BODIPY FL-C5-Ceramide. Data are presented with means ± standard deviations (SD) of values from three independent experiments. ***, P ≤ 0.001 (in comparison to controls, siScr and mock). Blots showing the efficacy the siRNA-mediated VAPA knockdowns are shown in .

    Article Snippet: The mouse anti-VAPA monoclonal antibody (MABN361) was from Millipore, the rabbit anti-CERT polyclonal antibody (PA5-115035) was from Invitrogen, the rabbit anti-ORP1L polyclonal antibody ORP1L (ab131165), the rat anti-nSMase2 (ab85017) and the rat anti-BrdU (ab6326) were from Abcam, the rabbit anti-GLTP (10850-1-AP) was from ThermoFisher, the mouse anti-phosphoglycan (Galβ1,4Manα1-PO4) CA7AE monoclonal antibody [ ] was from Cedarlane, the rabbit anti-β-actin polyclonal antibody was from Cell Signalling, and the rat LAMP-1 monoclonal antibody 1D4B developed by J.T.August and purchased through the Developmental Studies Hybridoma Bank at the University of lowa and the National Institute of Child Health and Human Development.

    Techniques: Transfection, Infection, Incubation, Comparison

    BMM were infected with L. amazonensis metacyclic promastigotes and at the indicated time points the localization of CERT (red) ( A ) and ORP1L (red) ( B ) was assessed by immunofluorescence confocal microscopy. Co-localization of VAPA (green) with CERT (red) ( A ) and ORP1L(red) ( B ) was assessed by immunofluorescence confocal microscopy at 72 h post-infection. DNA is shown in blue. White arrowheads denote internalized parasites. Representative images from three independent experiments are shown. BMM (normal, treated with siRNA to CERT or scramble siRNA) were infected with L. amazonensis metacyclic promastigotes and at the indicated time points post-infection, parasite burden ( C ) and PV size ( D ) were assessed. BMM (untreated, treated with siRNA to ORP1L or scramble siRNA) were infected with L. amazonensis metacyclic promastigotes and at the indicated time points post-infection parasite burden ( E ) and PV size ( F ) were assessed. Data are presented as the means ± SD of values from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001. For the determination of PV surface area ( D , F ) data are presented as clouds with means ± SD of values from three independent experiments for a total of 450 PVs. **, P ≤ 0.01; ***, P ≤ 0.001. Blots showing the efficacy the siRNA-mediated CERT and ORP1L knockdowns are shown in .

    Journal: PLOS Pathogens

    Article Title: VAPA mediates lipid exchange between Leishmania amazonensis and host macrophages

    doi: 10.1371/journal.ppat.1012636

    Figure Lengend Snippet: BMM were infected with L. amazonensis metacyclic promastigotes and at the indicated time points the localization of CERT (red) ( A ) and ORP1L (red) ( B ) was assessed by immunofluorescence confocal microscopy. Co-localization of VAPA (green) with CERT (red) ( A ) and ORP1L(red) ( B ) was assessed by immunofluorescence confocal microscopy at 72 h post-infection. DNA is shown in blue. White arrowheads denote internalized parasites. Representative images from three independent experiments are shown. BMM (normal, treated with siRNA to CERT or scramble siRNA) were infected with L. amazonensis metacyclic promastigotes and at the indicated time points post-infection, parasite burden ( C ) and PV size ( D ) were assessed. BMM (untreated, treated with siRNA to ORP1L or scramble siRNA) were infected with L. amazonensis metacyclic promastigotes and at the indicated time points post-infection parasite burden ( E ) and PV size ( F ) were assessed. Data are presented as the means ± SD of values from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001. For the determination of PV surface area ( D , F ) data are presented as clouds with means ± SD of values from three independent experiments for a total of 450 PVs. **, P ≤ 0.01; ***, P ≤ 0.001. Blots showing the efficacy the siRNA-mediated CERT and ORP1L knockdowns are shown in .

    Article Snippet: The mouse anti-VAPA monoclonal antibody (MABN361) was from Millipore, the rabbit anti-CERT polyclonal antibody (PA5-115035) was from Invitrogen, the rabbit anti-ORP1L polyclonal antibody ORP1L (ab131165), the rat anti-nSMase2 (ab85017) and the rat anti-BrdU (ab6326) were from Abcam, the rabbit anti-GLTP (10850-1-AP) was from ThermoFisher, the mouse anti-phosphoglycan (Galβ1,4Manα1-PO4) CA7AE monoclonal antibody [ ] was from Cedarlane, the rabbit anti-β-actin polyclonal antibody was from Cell Signalling, and the rat LAMP-1 monoclonal antibody 1D4B developed by J.T.August and purchased through the Developmental Studies Hybridoma Bank at the University of lowa and the National Institute of Child Health and Human Development.

    Techniques: Infection, Immunofluorescence, Confocal Microscopy

    L. amazonensis hijacks VAPA. BMMs were infected or not with either L. amazonensis (A-D), Δ lpg1 and Δ lpg1 + LPG1 L. donovani (E) or Δ gp63 and Δ gp63 + GP63 L. major (F) metacyclic promastigotes. At the indicated time points post-infection, in situ VAPA-CERT (A) and VAPA-ORP1L (C) complexes were detected by proximity ligation and visualized by confocal immunofluorescence microscopy (red dots). DNA is in blue. Representative images from 3 independent experiments are shown. Quantification of in situ complexes for VAPA-CERT (B) and VAPA-ORP1L (D) in uninfected BMM and in BMM infected with L. amazonensis . Quantification of in situ complexes for VAPA-ORP1L in uninfected BMM and in BMM infected with either (E) Δ lpg1 and Δ lpg1 + LPG1 L. donovani or (F) Δ gp63 and Δ gp63 + GP63 L. major . Data are presented as clouds with means ± standard deviations (SD) of values from three independent experiments for a total of 75 cells in each group. **, P ≤ 0.01; ***, P ≤ 0.001. Controls for the proximity ligation assays are shown in .

    Journal: PLOS Pathogens

    Article Title: VAPA mediates lipid exchange between Leishmania amazonensis and host macrophages

    doi: 10.1371/journal.ppat.1012636

    Figure Lengend Snippet: L. amazonensis hijacks VAPA. BMMs were infected or not with either L. amazonensis (A-D), Δ lpg1 and Δ lpg1 + LPG1 L. donovani (E) or Δ gp63 and Δ gp63 + GP63 L. major (F) metacyclic promastigotes. At the indicated time points post-infection, in situ VAPA-CERT (A) and VAPA-ORP1L (C) complexes were detected by proximity ligation and visualized by confocal immunofluorescence microscopy (red dots). DNA is in blue. Representative images from 3 independent experiments are shown. Quantification of in situ complexes for VAPA-CERT (B) and VAPA-ORP1L (D) in uninfected BMM and in BMM infected with L. amazonensis . Quantification of in situ complexes for VAPA-ORP1L in uninfected BMM and in BMM infected with either (E) Δ lpg1 and Δ lpg1 + LPG1 L. donovani or (F) Δ gp63 and Δ gp63 + GP63 L. major . Data are presented as clouds with means ± standard deviations (SD) of values from three independent experiments for a total of 75 cells in each group. **, P ≤ 0.01; ***, P ≤ 0.001. Controls for the proximity ligation assays are shown in .

    Article Snippet: The mouse anti-VAPA monoclonal antibody (MABN361) was from Millipore, the rabbit anti-CERT polyclonal antibody (PA5-115035) was from Invitrogen, the rabbit anti-ORP1L polyclonal antibody ORP1L (ab131165), the rat anti-nSMase2 (ab85017) and the rat anti-BrdU (ab6326) were from Abcam, the rabbit anti-GLTP (10850-1-AP) was from ThermoFisher, the mouse anti-phosphoglycan (Galβ1,4Manα1-PO4) CA7AE monoclonal antibody [ ] was from Cedarlane, the rabbit anti-β-actin polyclonal antibody was from Cell Signalling, and the rat LAMP-1 monoclonal antibody 1D4B developed by J.T.August and purchased through the Developmental Studies Hybridoma Bank at the University of lowa and the National Institute of Child Health and Human Development.

    Techniques: Infection, In Situ, Ligation, Immunofluorescence, Microscopy

    (A) BMM were infected with L. major metacyclic promastigotes and at 6 h post-phagocytosis, the localization of VAPA (green) and LPG (red) were assessed by confocal immunofluorescence microscopy. Colocalized pixels are in white and DNA is in blue. (B) BMM (untreated or treated with siRNA to VAPA, scrambled siRNA, or mock transfected) were infected with L. major metacyclic promastigotes and at 6 h post-phagocytosis, the localization of LPG (red) was assessed by confocal immunofluorescence microscopy. DNA is in blue. (C) . Untreated BMM (left panel) or BMM treated with siRNA to VAPA (right panel) were infected with L. major metacyclic promastigotes and at 6 h post-phagocytosis, the co-localization of LPG (red) with ERSeeing (green) or LAMP1 (green) was assessed by confocal immunofluorescence microscopy. Colocalized pixels are in white, DNA is in blue. White arrowheads denote internalized parasites. Representative images from 3 independent experiments are shown. Blots showing the efficacy the siRNA-mediated VAPA knockdowns are shown in .

    Journal: PLOS Pathogens

    Article Title: VAPA mediates lipid exchange between Leishmania amazonensis and host macrophages

    doi: 10.1371/journal.ppat.1012636

    Figure Lengend Snippet: (A) BMM were infected with L. major metacyclic promastigotes and at 6 h post-phagocytosis, the localization of VAPA (green) and LPG (red) were assessed by confocal immunofluorescence microscopy. Colocalized pixels are in white and DNA is in blue. (B) BMM (untreated or treated with siRNA to VAPA, scrambled siRNA, or mock transfected) were infected with L. major metacyclic promastigotes and at 6 h post-phagocytosis, the localization of LPG (red) was assessed by confocal immunofluorescence microscopy. DNA is in blue. (C) . Untreated BMM (left panel) or BMM treated with siRNA to VAPA (right panel) were infected with L. major metacyclic promastigotes and at 6 h post-phagocytosis, the co-localization of LPG (red) with ERSeeing (green) or LAMP1 (green) was assessed by confocal immunofluorescence microscopy. Colocalized pixels are in white, DNA is in blue. White arrowheads denote internalized parasites. Representative images from 3 independent experiments are shown. Blots showing the efficacy the siRNA-mediated VAPA knockdowns are shown in .

    Article Snippet: The mouse anti-VAPA monoclonal antibody (MABN361) was from Millipore, the rabbit anti-CERT polyclonal antibody (PA5-115035) was from Invitrogen, the rabbit anti-ORP1L polyclonal antibody ORP1L (ab131165), the rat anti-nSMase2 (ab85017) and the rat anti-BrdU (ab6326) were from Abcam, the rabbit anti-GLTP (10850-1-AP) was from ThermoFisher, the mouse anti-phosphoglycan (Galβ1,4Manα1-PO4) CA7AE monoclonal antibody [ ] was from Cedarlane, the rabbit anti-β-actin polyclonal antibody was from Cell Signalling, and the rat LAMP-1 monoclonal antibody 1D4B developed by J.T.August and purchased through the Developmental Studies Hybridoma Bank at the University of lowa and the National Institute of Child Health and Human Development.

    Techniques: Infection, Immunofluorescence, Microscopy, Transfection

    VAPA is required for the acquisition of host sphingolipids by L. amazonensis amastigotes, as well as for the transfer of the Leishmania virulence glycolipid lipophosphoglycan (LPG) to the host cell endoplasmic reticulum. The identity of the lipid transfer protein (s) (LTP) involved in this bi-directional lipid transfer remains to be determined. Created with BioRender.

    Journal: PLOS Pathogens

    Article Title: VAPA mediates lipid exchange between Leishmania amazonensis and host macrophages

    doi: 10.1371/journal.ppat.1012636

    Figure Lengend Snippet: VAPA is required for the acquisition of host sphingolipids by L. amazonensis amastigotes, as well as for the transfer of the Leishmania virulence glycolipid lipophosphoglycan (LPG) to the host cell endoplasmic reticulum. The identity of the lipid transfer protein (s) (LTP) involved in this bi-directional lipid transfer remains to be determined. Created with BioRender.

    Article Snippet: The mouse anti-VAPA monoclonal antibody (MABN361) was from Millipore, the rabbit anti-CERT polyclonal antibody (PA5-115035) was from Invitrogen, the rabbit anti-ORP1L polyclonal antibody ORP1L (ab131165), the rat anti-nSMase2 (ab85017) and the rat anti-BrdU (ab6326) were from Abcam, the rabbit anti-GLTP (10850-1-AP) was from ThermoFisher, the mouse anti-phosphoglycan (Galβ1,4Manα1-PO4) CA7AE monoclonal antibody [ ] was from Cedarlane, the rabbit anti-β-actin polyclonal antibody was from Cell Signalling, and the rat LAMP-1 monoclonal antibody 1D4B developed by J.T.August and purchased through the Developmental Studies Hybridoma Bank at the University of lowa and the National Institute of Child Health and Human Development.

    Techniques:

    ( A ) Left panel: Representative immunoblots showing the levels of VAPA and Tubulin in Control and VAPA KO cells. Tubulin expression level was used as loading control. Right panel: quantification of relative VAPA density in Control and VAPA KO cells normalized to Tubulin levels (mean ± SEM from three independent experiments). Data were analysed using a single-sample Student t-test. ( B ) Confocal images of Control and VAPA KO cells immunostained for VAPA. Scale bar 20 µm. ( C ) Confocal images of VAPA KO leader cells expressing wild-type mCherry-VAPA (VAPA KO +WT) or mCherry-VAPA KD/MD (VAPA KO +KDMD). Scale bar 10 µm. ( D ) Phase contrast images of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells migrating collectively 48 hr after space release. For each condition, the trajectory of a leader cell over 20 hr is shown in green. Scale bar: 100 µm. ( E ) Analysis of relative colonized surface within the last 20 h by Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells (mean ± SEM from three independent experiments). Data were analysed using a One-way Anova paired test. ( F, G ) Analysis of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cell velocity ( F ) n=40, 40, 3,9 and 39 cells respectively from three independent experiments and directionality coefficient ( G ) n=34, 36, 32, and 26 cells respectively from three independent experiments. Data were analysed using a One-way Anova Kruskal-Wallis test. ( H ) Phase contrast images of a Control and VAPA KO individual cell displacing on fibronectin-coated glass during 5 minutes. The cell trajectory is shown in blue. Scale bar: 20 µm. ( I, J ) Analysis of cell velocity ( I ) and directionality coefficient ( J ) of Control and VAPA KO individual cells displacing on fibronectin-coated glass during at least 3 h (mean ± SEM; Control: n=64 cells; VAPA KO: n=33 cells from two independent experiments). data were analysed using non parametric Mann-Whitney t-test. ( K ) Epifluorescence images of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells after 1 hour spreading on fibronectin-coated glass and stained as indicated. Scale bar: 50 µm. ( L ) Analysis of cells area after 1 hour spreading on fibronectin-coated glass (mean ± SEM; n=117, 134, 113, and 128 cells respectively from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test. (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 1—source data 1. Table containing the raw data used for the quantifications in . Figure 1—source data 2. Folder containing the 2 original files of the full raw unedited blots for VAPA and Tubulin presented in figure with the uncropped annotated blots.

    Journal: eLife

    Article Title: The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions

    doi: 10.7554/eLife.85962

    Figure Lengend Snippet: ( A ) Left panel: Representative immunoblots showing the levels of VAPA and Tubulin in Control and VAPA KO cells. Tubulin expression level was used as loading control. Right panel: quantification of relative VAPA density in Control and VAPA KO cells normalized to Tubulin levels (mean ± SEM from three independent experiments). Data were analysed using a single-sample Student t-test. ( B ) Confocal images of Control and VAPA KO cells immunostained for VAPA. Scale bar 20 µm. ( C ) Confocal images of VAPA KO leader cells expressing wild-type mCherry-VAPA (VAPA KO +WT) or mCherry-VAPA KD/MD (VAPA KO +KDMD). Scale bar 10 µm. ( D ) Phase contrast images of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells migrating collectively 48 hr after space release. For each condition, the trajectory of a leader cell over 20 hr is shown in green. Scale bar: 100 µm. ( E ) Analysis of relative colonized surface within the last 20 h by Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells (mean ± SEM from three independent experiments). Data were analysed using a One-way Anova paired test. ( F, G ) Analysis of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cell velocity ( F ) n=40, 40, 3,9 and 39 cells respectively from three independent experiments and directionality coefficient ( G ) n=34, 36, 32, and 26 cells respectively from three independent experiments. Data were analysed using a One-way Anova Kruskal-Wallis test. ( H ) Phase contrast images of a Control and VAPA KO individual cell displacing on fibronectin-coated glass during 5 minutes. The cell trajectory is shown in blue. Scale bar: 20 µm. ( I, J ) Analysis of cell velocity ( I ) and directionality coefficient ( J ) of Control and VAPA KO individual cells displacing on fibronectin-coated glass during at least 3 h (mean ± SEM; Control: n=64 cells; VAPA KO: n=33 cells from two independent experiments). data were analysed using non parametric Mann-Whitney t-test. ( K ) Epifluorescence images of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells after 1 hour spreading on fibronectin-coated glass and stained as indicated. Scale bar: 50 µm. ( L ) Analysis of cells area after 1 hour spreading on fibronectin-coated glass (mean ± SEM; n=117, 134, 113, and 128 cells respectively from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test. (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 1—source data 1. Table containing the raw data used for the quantifications in . Figure 1—source data 2. Folder containing the 2 original files of the full raw unedited blots for VAPA and Tubulin presented in figure with the uncropped annotated blots.

    Article Snippet: The following primary antibodies were used: mouse anti-EEA1 monoclonal antibody (BD transduction), rabbit anti-VAPB polyclonal antibody (Atlas antibody), mouse anti-VAPA monoclonal antibody and rabbit anti-VAPA polyclonal antibody for the immunoblot (Sigma), rabbit anti-TGN46 polyclonal antibody (Abcam), mouse anti-paxilline monoclonal antibody (Merck), mouse anti-cortactin monoclonal antibody (Merck), mouse anti-tubulin monoclonal antibody (GeneTex), mouse anti-PI(4,5)P2 monoclonal IgM antibody (Echelon).

    Techniques: Western Blot, Control, Expressing, MANN-WHITNEY, Staining

    ( A ) Left panel: representative immunoblots showing the levels of VAPB and Tubulin in Control and VAPA KO cells. Tubulin level was used as loading control. Right panel: quantification of relative VAPB density in Control and VAPA KO cells normalized to Tubulin levels (mean ± SEM from three independent experiments). Data were analysed using a single-sample Student t-test (ns: non significant). ( B ) Confocal images of Control and VAPA KO leader cells immunostained for VAPB. Scale bar: 20 µm (5 µm in insets). Figure 1—figure supplement 1—source data 1. Table containing the raw data used for the quantifications . Figure 1—figure supplement 1—source data 2. Folder containing the 2 original files of the full raw unedited blots for VAPB and Tubulin presented in and a figure with the uncropped annotated blots.

    Journal: eLife

    Article Title: The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions

    doi: 10.7554/eLife.85962

    Figure Lengend Snippet: ( A ) Left panel: representative immunoblots showing the levels of VAPB and Tubulin in Control and VAPA KO cells. Tubulin level was used as loading control. Right panel: quantification of relative VAPB density in Control and VAPA KO cells normalized to Tubulin levels (mean ± SEM from three independent experiments). Data were analysed using a single-sample Student t-test (ns: non significant). ( B ) Confocal images of Control and VAPA KO leader cells immunostained for VAPB. Scale bar: 20 µm (5 µm in insets). Figure 1—figure supplement 1—source data 1. Table containing the raw data used for the quantifications . Figure 1—figure supplement 1—source data 2. Folder containing the 2 original files of the full raw unedited blots for VAPB and Tubulin presented in and a figure with the uncropped annotated blots.

    Article Snippet: The following primary antibodies were used: mouse anti-EEA1 monoclonal antibody (BD transduction), rabbit anti-VAPB polyclonal antibody (Atlas antibody), mouse anti-VAPA monoclonal antibody and rabbit anti-VAPA polyclonal antibody for the immunoblot (Sigma), rabbit anti-TGN46 polyclonal antibody (Abcam), mouse anti-paxilline monoclonal antibody (Merck), mouse anti-cortactin monoclonal antibody (Merck), mouse anti-tubulin monoclonal antibody (GeneTex), mouse anti-PI(4,5)P2 monoclonal IgM antibody (Echelon).

    Techniques: Western Blot, Control

    ( A ) Confocal images with zoom boxes of central (green squares) and peripheral (blue squares) paxillin-labeled focal adhesions from Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cells in migrating monolayers 24 -48hr after insert removal. Scale bar: 20 µm (2 µm in insets). ( B, C ) Analysis of central ( B ) and peripheral ( C ) focal adhesion area quantified from images in A, in Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cells (Control: n=2968 central and 353 peripheral focal adhesions from 20 cells, VAPA KO: n=4329 central and 293 peripheral focal adhesions from 22 cells, VAPA KO +WT: n=3818 central and 336 peripheral focal adhesions from 24 cells, VAPA KO +KDMD: n=2801 central and 297 peripheral focal adhesions from 19 cells, from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test.( D ) Confocal images of actin cytoskeleton network in Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cells stained for cortactin and F-actin. Green arrows point to F-Actin transversal arcs. Plot profiles of the cortactin signal along the leading edge are shown on the right. Pink lines highlight the cortactin-rich protrusive subdomains. Scale bar: 20 µm.( E ) Differential Interference Contrast (DIC) images (left) and kymographs (right) along the yellow lines of Control and VAPA KO leader cells from a 30 min movie at 1 frame every 3 seconds, showing protrusion and retraction phases of the leading edge. Scale bar: 20 µm (left) and 5 µm (right). ( F, G ) Analysis of proportion of the leading edge enriched with cortactin ( F ) and mean size of cortactin-enriched domains at the leading edge normalized to the length of the leading edge ( G ) quantified from images in D (mean ± SEM; n=29 cells for each cell line, from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test. ( H ) Quantification of protrusion phases frequency per 30 min quantified from the kymographs in E, in Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cells (mean ± SEM; n=16, 21, 17 and 12 cells respectively, from three independent experiments). Data were analysed using a non parametric Mann-Whitney t-test. (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 2—source data 1. Table containing the raw data used for the quantifications .

    Journal: eLife

    Article Title: The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions

    doi: 10.7554/eLife.85962

    Figure Lengend Snippet: ( A ) Confocal images with zoom boxes of central (green squares) and peripheral (blue squares) paxillin-labeled focal adhesions from Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cells in migrating monolayers 24 -48hr after insert removal. Scale bar: 20 µm (2 µm in insets). ( B, C ) Analysis of central ( B ) and peripheral ( C ) focal adhesion area quantified from images in A, in Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cells (Control: n=2968 central and 353 peripheral focal adhesions from 20 cells, VAPA KO: n=4329 central and 293 peripheral focal adhesions from 22 cells, VAPA KO +WT: n=3818 central and 336 peripheral focal adhesions from 24 cells, VAPA KO +KDMD: n=2801 central and 297 peripheral focal adhesions from 19 cells, from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test.( D ) Confocal images of actin cytoskeleton network in Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cells stained for cortactin and F-actin. Green arrows point to F-Actin transversal arcs. Plot profiles of the cortactin signal along the leading edge are shown on the right. Pink lines highlight the cortactin-rich protrusive subdomains. Scale bar: 20 µm.( E ) Differential Interference Contrast (DIC) images (left) and kymographs (right) along the yellow lines of Control and VAPA KO leader cells from a 30 min movie at 1 frame every 3 seconds, showing protrusion and retraction phases of the leading edge. Scale bar: 20 µm (left) and 5 µm (right). ( F, G ) Analysis of proportion of the leading edge enriched with cortactin ( F ) and mean size of cortactin-enriched domains at the leading edge normalized to the length of the leading edge ( G ) quantified from images in D (mean ± SEM; n=29 cells for each cell line, from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test. ( H ) Quantification of protrusion phases frequency per 30 min quantified from the kymographs in E, in Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cells (mean ± SEM; n=16, 21, 17 and 12 cells respectively, from three independent experiments). Data were analysed using a non parametric Mann-Whitney t-test. (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 2—source data 1. Table containing the raw data used for the quantifications .

    Article Snippet: The following primary antibodies were used: mouse anti-EEA1 monoclonal antibody (BD transduction), rabbit anti-VAPB polyclonal antibody (Atlas antibody), mouse anti-VAPA monoclonal antibody and rabbit anti-VAPA polyclonal antibody for the immunoblot (Sigma), rabbit anti-TGN46 polyclonal antibody (Abcam), mouse anti-paxilline monoclonal antibody (Merck), mouse anti-cortactin monoclonal antibody (Merck), mouse anti-tubulin monoclonal antibody (GeneTex), mouse anti-PI(4,5)P2 monoclonal IgM antibody (Echelon).

    Techniques: Labeling, Control, Staining, MANN-WHITNEY

    ( A, B ) Confocal images and zoom boxes of PI(4)P distribution in Control and VAPA KO leader cells expressing GFP-PH-OSBP and immunostained for TGN46 ( A ) or EEA1 ( B ). Scale Bar: 10 µm. ( C ) Analysis of Golgi(TGN46)/cytosol (left panel) and Early endosomes(EEA1)/Cytosol (right panel) ratio of GFP-PH-OSBP signal, quantified from images in A and B, in Control and VAPA KO leader cells (Left panel: n=24–25 cells, Right panel: n=13 cells; from three independent experiments). ( D, H ) Top: Sum projection of confocal images of PI(4)P ( D ) and PI(4,5)P2 ( H ) distribution in Control and VAPA KO leader cells expressing mCherry-P4M SidM or RFP-PH-PLCδ1 respectively, represented as a color-coded heat map. Scale bar: 10 µm. Bottom: Plot profiles of normalized grey levels along the pink lines. ( E, I ) Analysis of PM/Cytosol ratio of mCherry-P4M SidM ( E ) and RFP-PH-PLCδ1 ( I ), quantified from plot profiles represented in D and H respectively, in protrusive domains at the leading edge of Control and VAPA KO leader cells (E: n=14–16 cells, I: n=26–27 cells; from three independent experiments). ( F ) XZ view of confocal images of Control and VAPA KO cells immunostained for PI(4,5)P2. Scale Bar: 5 µm. ( G ) Analysis of PI(4,5)P2 peak intensity, quantified from images in F, in Control and VAPA KO leader cells (Control: n=58 cells; VAPA KO: n=53 cells, from three independent experiments). All data were analysed using non parametric Mann-Whitney t-test (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 3—source data 1. Table containing the raw data used for the quantifications .

    Journal: eLife

    Article Title: The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions

    doi: 10.7554/eLife.85962

    Figure Lengend Snippet: ( A, B ) Confocal images and zoom boxes of PI(4)P distribution in Control and VAPA KO leader cells expressing GFP-PH-OSBP and immunostained for TGN46 ( A ) or EEA1 ( B ). Scale Bar: 10 µm. ( C ) Analysis of Golgi(TGN46)/cytosol (left panel) and Early endosomes(EEA1)/Cytosol (right panel) ratio of GFP-PH-OSBP signal, quantified from images in A and B, in Control and VAPA KO leader cells (Left panel: n=24–25 cells, Right panel: n=13 cells; from three independent experiments). ( D, H ) Top: Sum projection of confocal images of PI(4)P ( D ) and PI(4,5)P2 ( H ) distribution in Control and VAPA KO leader cells expressing mCherry-P4M SidM or RFP-PH-PLCδ1 respectively, represented as a color-coded heat map. Scale bar: 10 µm. Bottom: Plot profiles of normalized grey levels along the pink lines. ( E, I ) Analysis of PM/Cytosol ratio of mCherry-P4M SidM ( E ) and RFP-PH-PLCδ1 ( I ), quantified from plot profiles represented in D and H respectively, in protrusive domains at the leading edge of Control and VAPA KO leader cells (E: n=14–16 cells, I: n=26–27 cells; from three independent experiments). ( F ) XZ view of confocal images of Control and VAPA KO cells immunostained for PI(4,5)P2. Scale Bar: 5 µm. ( G ) Analysis of PI(4,5)P2 peak intensity, quantified from images in F, in Control and VAPA KO leader cells (Control: n=58 cells; VAPA KO: n=53 cells, from three independent experiments). All data were analysed using non parametric Mann-Whitney t-test (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 3—source data 1. Table containing the raw data used for the quantifications .

    Article Snippet: The following primary antibodies were used: mouse anti-EEA1 monoclonal antibody (BD transduction), rabbit anti-VAPB polyclonal antibody (Atlas antibody), mouse anti-VAPA monoclonal antibody and rabbit anti-VAPA polyclonal antibody for the immunoblot (Sigma), rabbit anti-TGN46 polyclonal antibody (Abcam), mouse anti-paxilline monoclonal antibody (Merck), mouse anti-cortactin monoclonal antibody (Merck), mouse anti-tubulin monoclonal antibody (GeneTex), mouse anti-PI(4,5)P2 monoclonal IgM antibody (Echelon).

    Techniques: Control, Expressing, MANN-WHITNEY

    ( A ) TIRF microscopy images of GFP-MAPPER foci distribution along the ER in Control and VAPA KO leader cells expressing GFP-MAPPER and RFP-KDEL. Scale bar: 5 µm. ( B ) Sequential TIRF microscopy images of ER and GFP-MAPPER foci accumulation at the front of a Control leader cell expressing GFP-MAPPER and RFP-KDEL. Scale bar: 1 µm. ( C ) Confocal images of GFP-MAPPER foci distribution along Cherry-VAPA containing ER in Control cells transiently expressing Cherry-VAPA. Scale bar: 1 µm. ( D ) Transmission Electron Microscopy images of transversal cuts of a Control leader cell, showing the leading edge. Arrows point to ER-PM contact sites at the bottom of the cell. Scale bar: 1 µm (top) and 100 nm in insets 1 and 2. ( E ) Sequential TIRF microscopy images of GFP-MAPPER foci at the front of Control and VAPA KO leader cells expressing GFP-MAPPER. Individual GFP-MAPPER foci at each time point are pictured in the frames below images. Scale bar: 1 µm. ( F, G ). Analysis of the lifetime ( F ) and the speed ( G ) of ventral GFP-MAPPER foci, quantified from images in E, in Control and VAPA KO leader cells (Control: n=92 and 52 foci from 8 cells; VAPA KO: n=77 and 58 foci from 6 cells, from four independent experiments). All data were analysed using non parametric Mann-Whitney t-test (***p-values <0.001). Figure 4—source data 1. Table containing the raw data used for the quantifications .

    Journal: eLife

    Article Title: The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions

    doi: 10.7554/eLife.85962

    Figure Lengend Snippet: ( A ) TIRF microscopy images of GFP-MAPPER foci distribution along the ER in Control and VAPA KO leader cells expressing GFP-MAPPER and RFP-KDEL. Scale bar: 5 µm. ( B ) Sequential TIRF microscopy images of ER and GFP-MAPPER foci accumulation at the front of a Control leader cell expressing GFP-MAPPER and RFP-KDEL. Scale bar: 1 µm. ( C ) Confocal images of GFP-MAPPER foci distribution along Cherry-VAPA containing ER in Control cells transiently expressing Cherry-VAPA. Scale bar: 1 µm. ( D ) Transmission Electron Microscopy images of transversal cuts of a Control leader cell, showing the leading edge. Arrows point to ER-PM contact sites at the bottom of the cell. Scale bar: 1 µm (top) and 100 nm in insets 1 and 2. ( E ) Sequential TIRF microscopy images of GFP-MAPPER foci at the front of Control and VAPA KO leader cells expressing GFP-MAPPER. Individual GFP-MAPPER foci at each time point are pictured in the frames below images. Scale bar: 1 µm. ( F, G ). Analysis of the lifetime ( F ) and the speed ( G ) of ventral GFP-MAPPER foci, quantified from images in E, in Control and VAPA KO leader cells (Control: n=92 and 52 foci from 8 cells; VAPA KO: n=77 and 58 foci from 6 cells, from four independent experiments). All data were analysed using non parametric Mann-Whitney t-test (***p-values <0.001). Figure 4—source data 1. Table containing the raw data used for the quantifications .

    Article Snippet: The following primary antibodies were used: mouse anti-EEA1 monoclonal antibody (BD transduction), rabbit anti-VAPB polyclonal antibody (Atlas antibody), mouse anti-VAPA monoclonal antibody and rabbit anti-VAPA polyclonal antibody for the immunoblot (Sigma), rabbit anti-TGN46 polyclonal antibody (Abcam), mouse anti-paxilline monoclonal antibody (Merck), mouse anti-cortactin monoclonal antibody (Merck), mouse anti-tubulin monoclonal antibody (GeneTex), mouse anti-PI(4,5)P2 monoclonal IgM antibody (Echelon).

    Techniques: Microscopy, Control, Expressing, Transmission Assay, Electron Microscopy, MANN-WHITNEY

    ( A ) Confocal images of GFP-MAPPER foci distribution along anti-VAPA-stained ER in Control cells. On the right: plot profiles of normalized gray values along the two lines depicted in yellow. Scale bar: 1 µm. ( B ) Sequential spinning disk images of dorsal (magenta) and ventral (green) GFP-MAPPER foci at the front of a Control leader cell expressing GFP-MAPPER. Top and bottom plane images were color-coded in magenta and green respectively. Circles highlight single GFP-MAPPER foci at the dorsal (blue) and ventral (red) sides. Scale bar: 3 µm. ( C ) Representative Transmission Electron Microscopy images of a Control and a VAPA KO cell showing ER-PM contact sites. Scale bar: 0.3 µm. The ER compartment and the PM are pictured in the right framebox. ( D, E ). Analysis of the ER portion in contact with the PM ( D ) and the percentage of the PM in contact with the ER ( E ), quantified from images in A, in Control and VAPA KO leader cells (mean ± SEM; Control: n=25 cells: VAPA KO: n=14 cells, from three independent experiments). All data were analysed using non parametric Mann-Whitney t-test (ns: non significant). Figure 4—figure supplement 1—source data 1. Table containing the raw data used for the quantifications and 1E.

    Journal: eLife

    Article Title: The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions

    doi: 10.7554/eLife.85962

    Figure Lengend Snippet: ( A ) Confocal images of GFP-MAPPER foci distribution along anti-VAPA-stained ER in Control cells. On the right: plot profiles of normalized gray values along the two lines depicted in yellow. Scale bar: 1 µm. ( B ) Sequential spinning disk images of dorsal (magenta) and ventral (green) GFP-MAPPER foci at the front of a Control leader cell expressing GFP-MAPPER. Top and bottom plane images were color-coded in magenta and green respectively. Circles highlight single GFP-MAPPER foci at the dorsal (blue) and ventral (red) sides. Scale bar: 3 µm. ( C ) Representative Transmission Electron Microscopy images of a Control and a VAPA KO cell showing ER-PM contact sites. Scale bar: 0.3 µm. The ER compartment and the PM are pictured in the right framebox. ( D, E ). Analysis of the ER portion in contact with the PM ( D ) and the percentage of the PM in contact with the ER ( E ), quantified from images in A, in Control and VAPA KO leader cells (mean ± SEM; Control: n=25 cells: VAPA KO: n=14 cells, from three independent experiments). All data were analysed using non parametric Mann-Whitney t-test (ns: non significant). Figure 4—figure supplement 1—source data 1. Table containing the raw data used for the quantifications and 1E.

    Article Snippet: The following primary antibodies were used: mouse anti-EEA1 monoclonal antibody (BD transduction), rabbit anti-VAPB polyclonal antibody (Atlas antibody), mouse anti-VAPA monoclonal antibody and rabbit anti-VAPA polyclonal antibody for the immunoblot (Sigma), rabbit anti-TGN46 polyclonal antibody (Abcam), mouse anti-paxilline monoclonal antibody (Merck), mouse anti-cortactin monoclonal antibody (Merck), mouse anti-tubulin monoclonal antibody (GeneTex), mouse anti-PI(4,5)P2 monoclonal IgM antibody (Echelon).

    Techniques: Staining, Control, Expressing, Transmission Assay, Electron Microscopy, MANN-WHITNEY

    ( A ) Sequential TIRF microscopy images of focal adhesions in Control and VAPA KO leader cells expressing mCherry-Vinculin. Scale Bar: 1 µm. ( B, C ) Analysis of assembly rate ( B ) and disassembly rate ( C ) of focal adhesions (FA), quantified from time-lapse images in A, in Control and VAPA KO leader cells (whisker plots with 10–90 percentile; Control: n=217 FA from 9 cells; VAPA KO: n=342 FA from 8 cells, from three independent experiments). Data were analysed using non parametric Mann-Whitney t-test. ( D ) Distribution of focal adhesion life times in Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cells (Control: n=98 FA from 10 cells; VAPA KO: n=120 FA from 12 cells, VAPA KO +WT: n=79 FA from 8 cells, VAPA KO +KDMD: n=90 FA from 9 cells, from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test. ( E ) Confocal images of focal adhesions after nocodazole treatment and wash-out in migrating Control and VAPA KO leader cells immunostained for paxillin. Scale bar: 10 µm (2 µm in insets). ( F ) Analysis of relative focal adhesions (FA) size, quantified from images in E, in Control and VAPA KO leader cells after 0 min, 15 min, 30 min, and 60 min after nocodazole wash-out (FA from 22 to 25 cells were analysed, from 3 independent experiments. Control T0min: n=6053 FA; Control T15 min: n=5146 FA; Control T30 min: n=5543 FA; Control T60 min: n=3913 FA; VAPA KO T0 min: n=4481 FA, VAPA KO T15 min: n=3878 FA, VAPA KO T30 min: n=4165 FA; T60 min: n=4889 FA). Data were analysed using non parametric Mann-Whitney t-test. (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 5—source data 1. Table containing the raw data used for the quantifications .

    Journal: eLife

    Article Title: The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions

    doi: 10.7554/eLife.85962

    Figure Lengend Snippet: ( A ) Sequential TIRF microscopy images of focal adhesions in Control and VAPA KO leader cells expressing mCherry-Vinculin. Scale Bar: 1 µm. ( B, C ) Analysis of assembly rate ( B ) and disassembly rate ( C ) of focal adhesions (FA), quantified from time-lapse images in A, in Control and VAPA KO leader cells (whisker plots with 10–90 percentile; Control: n=217 FA from 9 cells; VAPA KO: n=342 FA from 8 cells, from three independent experiments). Data were analysed using non parametric Mann-Whitney t-test. ( D ) Distribution of focal adhesion life times in Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cells (Control: n=98 FA from 10 cells; VAPA KO: n=120 FA from 12 cells, VAPA KO +WT: n=79 FA from 8 cells, VAPA KO +KDMD: n=90 FA from 9 cells, from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test. ( E ) Confocal images of focal adhesions after nocodazole treatment and wash-out in migrating Control and VAPA KO leader cells immunostained for paxillin. Scale bar: 10 µm (2 µm in insets). ( F ) Analysis of relative focal adhesions (FA) size, quantified from images in E, in Control and VAPA KO leader cells after 0 min, 15 min, 30 min, and 60 min after nocodazole wash-out (FA from 22 to 25 cells were analysed, from 3 independent experiments. Control T0min: n=6053 FA; Control T15 min: n=5146 FA; Control T30 min: n=5543 FA; Control T60 min: n=3913 FA; VAPA KO T0 min: n=4481 FA, VAPA KO T15 min: n=3878 FA, VAPA KO T30 min: n=4165 FA; T60 min: n=4889 FA). Data were analysed using non parametric Mann-Whitney t-test. (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 5—source data 1. Table containing the raw data used for the quantifications .

    Article Snippet: The following primary antibodies were used: mouse anti-EEA1 monoclonal antibody (BD transduction), rabbit anti-VAPB polyclonal antibody (Atlas antibody), mouse anti-VAPA monoclonal antibody and rabbit anti-VAPA polyclonal antibody for the immunoblot (Sigma), rabbit anti-TGN46 polyclonal antibody (Abcam), mouse anti-paxilline monoclonal antibody (Merck), mouse anti-cortactin monoclonal antibody (Merck), mouse anti-tubulin monoclonal antibody (GeneTex), mouse anti-PI(4,5)P2 monoclonal IgM antibody (Echelon).

    Techniques: Microscopy, Control, Expressing, Whisker Assay, MANN-WHITNEY

    ( A ) TIRF microscopy images of GFP-MAPPER and focal adhesions in Control and VAPA KO leader cells expressing GFP-MAPPER and mCherry-Vinculin. Scale bars: 10 µm (1 µm in insets). ( B ) Analysis of the percentage of central (top) or peripheral (bottom) focal adhesions in contact with GFP-MAPPER foci in Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cells (n=10–12 cells, from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test. ( C ) Sequential TIRF microscopy images of GFP-MAPPER foci and focal adhesions before and after its disassembly in a Control and 2 representative VAPA KO leader cells expressing GFP-MAPPER and mCherry-Vinculin. Scale bar: 1 µm. ( D ) Time course of GFP-MAPPER (green) and mCherry-Vinculin (magenta) signals during the lifetime of focal adhesions in the yellow ROI depicted in C. The signals were smoothed, readjusted to the minimal value and expressed as % of the maximal value. ( E ) Histogram representing the repartition of first anchoring time of GFP-MAPPER foci relative to focal adhesion disassembly in Control and VAPA KO leader cells, quantified from images in D (n=21 focal adhesions from 7 cells from three independent experiments). ( F ) Analysis of duration of the first anchoring of GFP-MAPPER foci to focal adhesions in Control and VAPA KO leader cells, from images in D (n=21 focal adhesions from 7 cells from three independent experiments). Data were analysed using a non parametric Mann-Whitney t-test. ( G ) TIRF microscopy images of iRFP-Vinculin, GFP-MAPPER and Cherry-VAPA WT in a VAPA KO leader cell. Scale bar: 2 µm. ( H ) Plot profiles of normalized grey levels along the two lines depicted in G. (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 6—source data 1. Table containing the raw data used for the quantifications .

    Journal: eLife

    Article Title: The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions

    doi: 10.7554/eLife.85962

    Figure Lengend Snippet: ( A ) TIRF microscopy images of GFP-MAPPER and focal adhesions in Control and VAPA KO leader cells expressing GFP-MAPPER and mCherry-Vinculin. Scale bars: 10 µm (1 µm in insets). ( B ) Analysis of the percentage of central (top) or peripheral (bottom) focal adhesions in contact with GFP-MAPPER foci in Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cells (n=10–12 cells, from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test. ( C ) Sequential TIRF microscopy images of GFP-MAPPER foci and focal adhesions before and after its disassembly in a Control and 2 representative VAPA KO leader cells expressing GFP-MAPPER and mCherry-Vinculin. Scale bar: 1 µm. ( D ) Time course of GFP-MAPPER (green) and mCherry-Vinculin (magenta) signals during the lifetime of focal adhesions in the yellow ROI depicted in C. The signals were smoothed, readjusted to the minimal value and expressed as % of the maximal value. ( E ) Histogram representing the repartition of first anchoring time of GFP-MAPPER foci relative to focal adhesion disassembly in Control and VAPA KO leader cells, quantified from images in D (n=21 focal adhesions from 7 cells from three independent experiments). ( F ) Analysis of duration of the first anchoring of GFP-MAPPER foci to focal adhesions in Control and VAPA KO leader cells, from images in D (n=21 focal adhesions from 7 cells from three independent experiments). Data were analysed using a non parametric Mann-Whitney t-test. ( G ) TIRF microscopy images of iRFP-Vinculin, GFP-MAPPER and Cherry-VAPA WT in a VAPA KO leader cell. Scale bar: 2 µm. ( H ) Plot profiles of normalized grey levels along the two lines depicted in G. (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 6—source data 1. Table containing the raw data used for the quantifications .

    Article Snippet: The following primary antibodies were used: mouse anti-EEA1 monoclonal antibody (BD transduction), rabbit anti-VAPB polyclonal antibody (Atlas antibody), mouse anti-VAPA monoclonal antibody and rabbit anti-VAPA polyclonal antibody for the immunoblot (Sigma), rabbit anti-TGN46 polyclonal antibody (Abcam), mouse anti-paxilline monoclonal antibody (Merck), mouse anti-cortactin monoclonal antibody (Merck), mouse anti-tubulin monoclonal antibody (GeneTex), mouse anti-PI(4,5)P2 monoclonal IgM antibody (Echelon).

    Techniques: Microscopy, Control, Expressing, MANN-WHITNEY