gfp vap b  (Addgene inc)

Journal: bioRxiv

Article Title: Selective MOSPD2-STARD3 interaction at ER contact sites governs late endosome/lysosome dynamics and cholesterol homeostasis

doi: 10.64898/2026.03.30.714413

Figure Lengend Snippet: A-B: Immunoprecipitation (anti-Flag) experiments between Flag-STARD3 and GFP-VAP-A, GFP-VAP-B, GFP-MOSPD2, GFP-MOSPD2 RD/LD, and GFP-VAP-A KD/MD (B) in HeLa cells. Approximatively 5 µg of total protein extract was analyzed by Western blot using anti-GFP, anti-STARD3, and anti-GAPDH antibodies. Immunoprecipitated proteins were analyzed using anti-GFP and anti-STARD3 antibodies. C: Immunoprecipitation between endogenous STARD3 and VAP-A, VAP-B and MOSPD2 in HCC1954 cells. Immunoprecipitation was performed using control IgG or anti-STARD3 antibodies in triplicate. Total protein extracts and immunoprecipitated proteins were analyzed by Western blot using anti-MOSPD2, anti-VAP-A, anti-VAP-B, anti-STARD3, and anti-GAPDH antibodies. *: aspecific. D: Principle of the native Holdup assay. Total protein extracts (a) are incubated with streptavidin resin saturated with a biotinylated MSP domain or control resin (b). After reaching equilibrium, unbound proteins are filtered out and quantified by Western blot. Binding intensity = 1 – (C Unbound / C total ). E: Coomassie blue staining of recombinant proteins used for native Holdup experiments: MBP alone or fused to the MSP domains of VAP-A, VAP-B or MOSPD2 (WT and RD/LD mutant), tagged with a 6 His for purification and biotinylated thanks to an AviTag. A total of 25 pmol of each protein was loaded. F: Native Holdup experiments quantifying the interaction between the recombinant MSP domains of VAP-A, VAP-B, MOSPD2, and MOSPD2 RD/LD and endogenous STARD3. Left: western blot analysis of the unbound prey protein (STARD3) in HCC1954 protein extracts after incubation with increasing amounts of the recombinant MSP domains. Right: Binding intensity between the MSP domains and the prey protein (STARD3). Binding curves were fitted using a Hill equation (mean ± SEM from 2 technical replicates), and apparent affinities ( K app ) and maximal binding intensities ( B max ) were calculated (± SD). G: FRAP experiment in HeLa cells co-expressing mCherry-STARD3 and either GFP-VAP-A, GFP-VAP-B, or GFP-MOSPD2. a: images showing STARD3-positive LE/Lys in close apposition to ER-localized GFP-MOSPD2 (top), GFP-VAP-A (middle), or GFP-VAP-B (bottom). Left: colocalization of mCherry-STARD3 (magenta) and GFP (green) pre-bleach. GFP signal (gray) displayed sequentially from left to right: pre-bleach, immediately post-bleach, 3 seconds post-bleach, and 20 s post-bleach. b: Quantification of relative GFP-signal intensity in the bleached ROI during the 2 seconds before bleaching, and the 21 seconds following bleaching in cells expressing GFP-VAP-A (blue curve), GFP-VAP-B (green curve), and GFP-MOSPD2 (red curve). The gray curve represents the GFP signal in the absence of bleaching. Mean values and standard deviations (black bars) are shown. The calculated half-times of recovery (mean t½ ± SD) are indicated.

Article Snippet: The pQCXIP MOSPD2 (WT; RD/LD; ΔCRAL-TRIO; ΔMSP), pQCXIP GFP-MOSPD2 (WT: Addgene #186467; RD/LD #186468), pQCXIP mScarlet-MOSPD2 (WT: Addgene #186472; RD/LD Addgene #186476; W201E: Addgene plasmid #186477; ΔMSP; ΔCRAL-TRIO mutants), GFP-VAP-A (WT: Addgene #104447; KD/MD Addgene #104449), GFP-VAP-B (Addgene plasmid #104448), EGFP-ER[TM(SAC1)] (Addgene plasmid #186475), pQCXIP mScarlet-ER [TM(SAC1)] (Addgene plasmid # 186572), Flag-STARD3, GFP-VAP-A (WT and KD/MD mutant), and GFP-VAP-B, expression vectors were previously described ( , ; ; ; ).

Techniques: Immunoprecipitation, Western Blot, Control, Incubation, Binding Assay, Staining, Recombinant, Mutagenesis, Purification, Expressing

Journal: bioRxiv

Article Title: Selective MOSPD2-STARD3 interaction at ER contact sites governs late endosome/lysosome dynamics and cholesterol homeostasis

doi: 10.64898/2026.03.30.714413

Figure Lengend Snippet: A-B: Immunoprecipitation (anti-Flag) experiments between Flag-STARD3 and GFP-VAP-A, GFP-VAP-B, GFP-MOSPD2, GFP-MOSPD2 RD/LD, and GFP-VAP-A KD/MD (B) in HeLa cells. Approximatively 5 µg of total protein extract was analyzed by Western blot using anti-GFP, anti-STARD3, and anti-GAPDH antibodies. Immunoprecipitated proteins were analyzed using anti-GFP and anti-STARD3 antibodies. C: Immunoprecipitation between endogenous STARD3 and VAP-A, VAP-B and MOSPD2 in HCC1954 cells. Immunoprecipitation was performed using control IgG or anti-STARD3 antibodies in triplicate. Total protein extracts and immunoprecipitated proteins were analyzed by Western blot using anti-MOSPD2, anti-VAP-A, anti-VAP-B, anti-STARD3, and anti-GAPDH antibodies. *: aspecific. D: Principle of the native Holdup assay. Total protein extracts (a) are incubated with streptavidin resin saturated with a biotinylated MSP domain or control resin (b). After reaching equilibrium, unbound proteins are filtered out and quantified by Western blot. Binding intensity = 1 – (C Unbound / C total ). E: Coomassie blue staining of recombinant proteins used for native Holdup experiments: MBP alone or fused to the MSP domains of VAP-A, VAP-B or MOSPD2 (WT and RD/LD mutant), tagged with a 6 His for purification and biotinylated thanks to an AviTag. A total of 25 pmol of each protein was loaded. F: Native Holdup experiments quantifying the interaction between the recombinant MSP domains of VAP-A, VAP-B, MOSPD2, and MOSPD2 RD/LD and endogenous STARD3. Left: western blot analysis of the unbound prey protein (STARD3) in HCC1954 protein extracts after incubation with increasing amounts of the recombinant MSP domains. Right: Binding intensity between the MSP domains and the prey protein (STARD3). Binding curves were fitted using a Hill equation (mean ± SEM from 2 technical replicates), and apparent affinities ( K app ) and maximal binding intensities ( B max ) were calculated (± SD). G: FRAP experiment in HeLa cells co-expressing mCherry-STARD3 and either GFP-VAP-A, GFP-VAP-B, or GFP-MOSPD2. a: images showing STARD3-positive LE/Lys in close apposition to ER-localized GFP-MOSPD2 (top), GFP-VAP-A (middle), or GFP-VAP-B (bottom). Left: colocalization of mCherry-STARD3 (magenta) and GFP (green) pre-bleach. GFP signal (gray) displayed sequentially from left to right: pre-bleach, immediately post-bleach, 3 seconds post-bleach, and 20 s post-bleach. b: Quantification of relative GFP-signal intensity in the bleached ROI during the 2 seconds before bleaching, and the 21 seconds following bleaching in cells expressing GFP-VAP-A (blue curve), GFP-VAP-B (green curve), and GFP-MOSPD2 (red curve). The gray curve represents the GFP signal in the absence of bleaching. Mean values and standard deviations (black bars) are shown. The calculated half-times of recovery (mean t½ ± SD) are indicated.

Article Snippet: The pQCXIP MOSPD2 (WT; RD/LD; ΔCRAL-TRIO; ΔMSP), pQCXIP GFP-MOSPD2 (WT: Addgene #186467; RD/LD #186468), pQCXIP mScarlet-MOSPD2 (WT: Addgene #186472; RD/LD Addgene #186476; W201E: Addgene plasmid #186477; ΔMSP; ΔCRAL-TRIO mutants), GFP-VAP-A (WT: Addgene #104447; KD/MD Addgene #104449), GFP-VAP-B (Addgene plasmid #104448), EGFP-ER[TM(SAC1)] (Addgene plasmid #186475), pQCXIP mScarlet-ER [TM(SAC1)] (Addgene plasmid # 186572), Flag-STARD3, GFP-VAP-A (WT and KD/MD mutant), and GFP-VAP-B, expression vectors were previously described ( , ; ; ; ).

Techniques: Immunoprecipitation, Western Blot, Control, Incubation, Binding Assay, Staining, Recombinant, Mutagenesis, Purification, Expressing

Journal: bioRxiv

Article Title: Selective MOSPD2-STARD3 interaction at ER contact sites governs late endosome/lysosome dynamics and cholesterol homeostasis

doi: 10.64898/2026.03.30.714413

Figure Lengend Snippet: A-F: Confocal images of WT HeLa cells (A) and MOSPD2-deficient HeLa cells (B-F), either untransfected (A-B) or transfected with expression vectors encoding WT STARD3 (C), the STARD3 SD/PA mutant (D), the STARD3 S209A mutant (E), or GFP-VAP-A (F). LE/Lys were labeled with anti-LAMP1 antibodies (magenta), and STARD3 was detected using anti-STARD3 antibodies. G-H: Quantification of LE/Lys numbers (G) and sizes (H) under the conditions shown in panels A-F. Data are displayed as Superplots showing the mean per cell (small dots) and per independent experiment (large dots). Independent experiments (n = 3-4) are color-coded. Means and error bars (SD) are shown as black bars. Data were collected from 108 (WT), 95 (KO MOSPD2 NT), 86 (KO MOSPD2 + STARD3 WT), 84 (KO MOSPD2 + STARD3 SD/PA), 86 (KO MOSPD2 + STARD3 S209A), and 48 (KO MOSPD2 + VAP-A) cells. One-way ANOVA with Tukey’s multiple comparisons test (ns, not significant; ****, P < 0.0001; n = 3-4 independent experiments).

Article Snippet: The pQCXIP MOSPD2 (WT; RD/LD; ΔCRAL-TRIO; ΔMSP), pQCXIP GFP-MOSPD2 (WT: Addgene #186467; RD/LD #186468), pQCXIP mScarlet-MOSPD2 (WT: Addgene #186472; RD/LD Addgene #186476; W201E: Addgene plasmid #186477; ΔMSP; ΔCRAL-TRIO mutants), GFP-VAP-A (WT: Addgene #104447; KD/MD Addgene #104449), GFP-VAP-B (Addgene plasmid #104448), EGFP-ER[TM(SAC1)] (Addgene plasmid #186475), pQCXIP mScarlet-ER [TM(SAC1)] (Addgene plasmid # 186572), Flag-STARD3, GFP-VAP-A (WT and KD/MD mutant), and GFP-VAP-B, expression vectors were previously described ( , ; ; ; ).

Techniques: Transfection, Expressing, Mutagenesis, Labeling

Journal: The Journal of Biological Chemistry

Article Title: VAMP-associated protein-A and oxysterol-binding protein–related protein 3 promote the entry of late endosomes into the nucleoplasmic reticulum

doi: 10.1074/jbc.RA118.003725

Figure Lengend Snippet: VAP-A is associated with type II NEI containing late endosomes. A and B, VAP-A–GFP-transfected FEMX-I cells were immunolabeled for SUN2 (A) or infected with Rab7–RFP baculovirus (B) and analyzed by CLSM. Area of a cross-section of NEI is magnified. A single x-y optical section is presented. C, FEMX-I cells expressing VAP-A–GFP and Rab7–RFP were analyzed by time-lapse video microscopy. Elapsed time is indicated in the top right corner. Arrowheads show the localization of VAP-A–GFP in SUN2-immunolabeled NEI (A), and arrows indicate Rab7–RFP+ late endosomes in NEI (B and C). Cartoon illustrates the direction (arrow) of Rab7+ late endosomes (LE) in NEI. D, cells expressing ER–GFP marker were double-immunolabeled for VAP-A and SUN2. A three-dimensional (3D) reconstruction of two adjacent sections (0.4-μm each) is presented. Only part of the nucleus is shown. Note the absence (purple arrow) or presence (yellow arrows) of VAP-A and ER–GFP in SUN2+ type I and II NEI, respectively. Additional information is provided in Fig. S1. E, percentage of SUN2+ NEI (type II) containing VAP-A or ER–GFP is presented. The means ± S.D. are shown (n = 3). The average of each experiment, where more than 50 cells were evaluated, is indicated. F, EM of FEMX-I cells shows that type II NEI are not only superficial indentations of the nuclear envelope but also appeared as deep recesses (yellow arrows). G and H, immunogold labeling on ultrathin cryosections reveals the presence of VAP-A–GFP in deep NEI of FEMX-I cells expressing the fusion protein (G, white arrowheads), whereas Rab7 proteins (H, black arrowheads) are found in the cytoplasm (left panel) and NEI (right panel) of FEMX-I cells. The presence of membrane-bound organelles in NEI is indicated with red arrowheads. nu, nucleoplasm. Scale bars, 5 μm (A–D) or as indicated (F–H).

Article Snippet: Transfection FEMX-I cells were transfected with 10 μg of CD9–GFP plasmid using FuGENE® HD transfection reagents (Promega, Madison, WI) or VAP-A–GFP plasmid with Lipofectamine 3000 compounds (ThermoFisher Scientific).

Techniques: Transfection, Immunolabeling, Infection, Expressing, Microscopy, Marker, Labeling

Journal: The Journal of Biological Chemistry

Article Title: VAMP-associated protein-A and oxysterol-binding protein–related protein 3 promote the entry of late endosomes into the nucleoplasmic reticulum

doi: 10.1074/jbc.RA118.003725

Figure Lengend Snippet: VAP-A is required for the entry of Rab7+ late endosomes in NEI. A–H, untransfected FEMX-I cells (control) or stably transfected with plasmids carrying scrambled shRNA, shVAP-A, or shVAP-B were analyzed by immunoblotting for VAP-A, VAP-B, and β-actin (A and E). Molecular mass markers (kDa) are indicated. Representative blots are shown. The relative VAP-A or VAP-B expression was quantified by comparison with control samples (B and F, red dotted line). The samples were normalized to β-actin. The means ± S.D. is shown with the individual value of each experience (n = 3). Alternatively, scrambled shRNA or shVAP-A/B transfected cells were infected with Rab7–RFP baculovirus and analyzed by CLSM after double-immunolabeling for VAP-A (C and D) or VAP-B (G and H) and SUN2. 3D reconstruction of 2–3 adjacent sections (0.4 μm each) is presented. Arrows indicate the presence of a given marker in NEI. Percentage of cells harboring either SUN2+ NEI-associated Rab7+ late endosomes, SUN2+ NEI only, or without NEI upon silencing VAP-A (D, see also Fig. S3) or VAP-B (H) were quantified. The means ± S.D. (D and H) are shown (n = 3). More than 50 cells were evaluated per experiment. p values are indicated. n.s., not significant. Images depicted in C are displayed in Videos S2 and S3. I, scrambled shRNA or shVAP-A–transfected FEMX-I cells were infected with ER–GFP and Rab7–RFP baculoviruses and analyzed by time-lapse video microscopy. Single x-y optical section (0.4 um) is presented. Elapsed time is indicated in the top right corner. Red and white arrows show the localization of Rab7–RFP in two distinct ER–GFP+ NEI (top, transverse section of NEI), and arrowheads indicate NEI without Rab7–RFP (bottom, sagittal section of three NEI). nu, nucleoplasm. Scale bars, 5 μm.

Article Snippet: Transfection FEMX-I cells were transfected with 10 μg of CD9–GFP plasmid using FuGENE® HD transfection reagents (Promega, Madison, WI) or VAP-A–GFP plasmid with Lipofectamine 3000 compounds (ThermoFisher Scientific).

Techniques: Stable Transfection, Transfection, shRNA, Western Blot, Expressing, Infection, Immunolabeling, Marker, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: VAMP-associated protein-A and oxysterol-binding protein–related protein 3 promote the entry of late endosomes into the nucleoplasmic reticulum

doi: 10.1074/jbc.RA118.003725

Figure Lengend Snippet: ORP3 is essential for the entry of Rab7+ late endosomes in NEI and co-localized with VAP-A and Rab7. A and B, FEMX-I cells stably transfected with plasmids carrying scrambled shRNA or shVAP-A were immunolabeled for ORP3 and SUN2 prior to CLSM. Single x-y optical section is presented. The amount of SUN2+ NEI containing ORP3 (A, square, see insets at right) was quantified (B). C and D, scrambled shRNA- or shORP3-transfected cells were infected with Rab7–RFP baculovirus and analyzed by CLSM after double-immunolabeling for ORP3 and SUN2 (C). Percentage of cells harboring either SUN2+ NEI-associated Rab7+ late endosomes, SUN2+ NEI only, or without NEI upon silencing ORP3 were quantified (D). The means ± S.D. are shown (n = 3). More than 30 cells were evaluated per experiment, and their average is presented (B). p values are indicated. E and F, FEMX-I cells expressing VAP-A–GFP were double-immunolabeled for ORP3 and Rab7 prior to CLSM. Rab7 antibody was directly conjugated to fluorophore Alexa Fluor 647, and an anti-mouse secondary antibody coupled to TRITC was used to detect ORP3 (E). The inset shows the merged panel with a 4.0-μm-long arrow indicating the area subjected to line scan analysis with RFI of each channel (F). Thick line indicates the position of late endosomes in NEI where three fluorescent signals overlaid. In each panel, arrows show the localization of a given protein in NEI. Images depicted in C (top panels) are displayed in Video S4. nu, nucleoplasm. Scale bars, 5 μm.

Article Snippet: Transfection FEMX-I cells were transfected with 10 μg of CD9–GFP plasmid using FuGENE® HD transfection reagents (Promega, Madison, WI) or VAP-A–GFP plasmid with Lipofectamine 3000 compounds (ThermoFisher Scientific).

Techniques: Stable Transfection, Transfection, shRNA, Immunolabeling, Infection, Expressing

Journal: The Journal of Biological Chemistry

Article Title: VAMP-associated protein-A and oxysterol-binding protein–related protein 3 promote the entry of late endosomes into the nucleoplasmic reticulum

doi: 10.1074/jbc.RA118.003725

Figure Lengend Snippet: Tripartite complex of VAP-A, ORP3, and Rab7 in NEI. A and B, detergent lysates prepared from transfected FEMX-I cells expressing VAP-A–GFP (VAP-A–GFP+ FEMX-I) or untransfected cells (FEMX-I) were subjected to immunoisolation (IS) with either anti-GFP antibody-coupled magnetic beads or anti-ORP3 (A) or anti-VAP-B (B) antibody followed by protein G-coupled magnetic beads as indicated above each lane. An aliquot of the input (1:50) and the entire bound fractions were probed for ORP3, VAP-A/B, and Rab7 by immunoblotting. Molecular mass markers (kDa) are indicated. The antibody used is indicated in the top left corner of the blot. Arrows indicate the protein of interest, and the arrowhead indicates VAP-A–GFP protein. Representative blots are shown (n = 3–6). Note that the VAP-A–GFP expression level is similar to endogenous protein in VAP-A–GFP+ FEMX-I cells, and almost no VAP-B is co-immunoisolated with ORP3 in contrast to VAP-A.

Article Snippet: Transfection FEMX-I cells were transfected with 10 μg of CD9–GFP plasmid using FuGENE® HD transfection reagents (Promega, Madison, WI) or VAP-A–GFP plasmid with Lipofectamine 3000 compounds (ThermoFisher Scientific).

Techniques: Transfection, Expressing, Magnetic Beads, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: VAMP-associated protein-A and oxysterol-binding protein–related protein 3 promote the entry of late endosomes into the nucleoplasmic reticulum

doi: 10.1074/jbc.RA118.003725

Figure Lengend Snippet: FRET analysis of the interaction between ORP3, VAP-A/B, and Rab7 in NEI. A–F, potential interactions between ORP3, VAP proteins, and Rab7 were evaluated by FRET using the acceptor photobleaching method. When appropriate, FEMX-I cells expressing VAP-A–GFP and/or Rab7–RFP were immunolabeled for ORP3 or VAP-B followed by secondary antibody coupled to appropriate fluorophore (TRITC or FITC). Intensity profiles and FRET efficiencies of each pair in NEI (A–D) or cytoplasm (E and F) are presented. The means ± S.D. are shown (n = 3). At least five cells were evaluated per experiment, and all of them are plotted. Acceptor photobleaching with a 561-nm laser line starts at time 15 s. FRET efficiencies were measured in a given ROI in bleached and unbleached areas. Corresponding micrographs with ROI are shown in Fig. S7.

Article Snippet: Transfection FEMX-I cells were transfected with 10 μg of CD9–GFP plasmid using FuGENE® HD transfection reagents (Promega, Madison, WI) or VAP-A–GFP plasmid with Lipofectamine 3000 compounds (ThermoFisher Scientific).

Techniques: Expressing, Immunolabeling

Journal: The Journal of Biological Chemistry

Article Title: VAMP-associated protein-A and oxysterol-binding protein–related protein 3 promote the entry of late endosomes into the nucleoplasmic reticulum

doi: 10.1074/jbc.RA118.003725

Figure Lengend Snippet: VOR complex is required for nuclear transfer of EV-derived proteins and nucleic acids. A and B, FEMX-I cells expressing either VAP-A–GFP (A) or ER–RFP (B) were incubated for 5 h with DiI-labeled EVs or CD9–GFP EVs, respectively, prior to analysis by time-lapse video microscopy. Elapsed time is indicated in the top right corner. Arrows indicate the entry of late endosomes containing DiI-labeled membranes (A) or CD9–GFP (B) in NEI and the arrowhead their tether to VAP-A–GFP+ ONM (A). GFP+ signals appearing in the nucleoplasm were highlighted (B, circles). Inset in B shows enlargement of NEI (dashed line) containing discrete punctate CD9–GFP signals. Still images (A) are from Video S5. C–E, scrambled shRNA (control, Ctl) or shVAP-A/B–transfected FEMX-I (C and D) or HeLa (E) cells were incubated with fluorescent EVs derived from CD9–GFP-expressing FEMX-I cells and then double-immunolabeled for VAP-A or VAP-B and SUN2 prior to CLSM. The amount of EV-derived CD9–GFP in the nuclear compartment was quantified using Fiji software. Micrographs are presented in Fig. S8. Independent values for each cell from three independent experiments (C, #1–3) and their average from three independent experiments (D and E) are presented. F and G, nuclei of FEMX-I cells incubated with CD9–GFP+ EVs were isolated before SUN2 immunolabeling and CLSM. Composite, single x-y section and z-projections are shown (F). GFP+ signals in the nucleoplasm were highlighted (circles) and quantified (G). H, scheme of isolation of EVs from FEMX-I cells and subsequent nucleic acid staining with SYTO 64 dye. I and J, scrambled shRNA or shVAP-A–transfected FEMX-I cells were incubated with SYTO 64-labeled EVs and immunolabeled as above. Nuclear SYTO 64 signals in a given cell (I, experiment #1) and their average from three independent experiments (J) were quantified. In all cases, means ± S.D. are shown (n = 3). More than 50 (C and D) and 30 (E, I, and J) cells or 30 isolated nuclei (G) were evaluated per experiment. p values are indicated. n.s., not significant. Scale bars, 5 μm.

Article Snippet: Transfection FEMX-I cells were transfected with 10 μg of CD9–GFP plasmid using FuGENE® HD transfection reagents (Promega, Madison, WI) or VAP-A–GFP plasmid with Lipofectamine 3000 compounds (ThermoFisher Scientific).

Techniques: Derivative Assay, Expressing, Incubation, Labeling, Microscopy, shRNA, Transfection, Immunolabeling, Software, Isolation, Staining