gfp vap b (Addgene inc)

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Addgene inc gfp vap b

A-B: Immunoprecipitation (anti-Flag) experiments between Flag-STARD3 and GFP-VAP-A, <t>GFP-VAP-B,</t> 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.

Gfp Vap B, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pegfpc1+hvap+a+kd+md/pEGFPC1-hVAP-A+KD%2FMD+(Plasmid+%23104449)/bio_rxiv__64898__2026__03__30__714413-271-36-37
Average 94 stars, based on 6 article reviews

gfp vap b - by Bioz Stars, 2026-09

94/100 stars

Images

1) Product Images from "Selective MOSPD2-STARD3 interaction at ER contact sites governs late endosome/lysosome dynamics and cholesterol homeostasis"

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

Journal: bioRxiv

doi: 10.64898/2026.03.30.714413

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.

Figure Legend 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.

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

Retroviral:

Article Title: STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch
Article Snippet: The GFP-MOSPD2 (WT Addgene plasmid # 186467; http://n2t.net/addgene:186467 ; RRID:Addgene_186467 and RD/LD mutant Addgene plasmid # 186468; http://n2t.net/addgene:186468 ; RRID:Addgene_186468), and Flag-tagged STARD3 expression vectors were previously described ( ; ). .. The retroviral expression vector pQCXIP encoding GFP-VAP-A (WT and KD/MD mutant) and GFP-VAP-B (WT and KD/MD mutant) were constructed by cloning PCR-amplified fragments from pEGFPC1-hVAP-A (Addgene plasmid # 104447), pEGFPC1-hVAP-A KD/MD (Addgene plasmid # 104449), pEGFPC1-hVAP-B (Addgene plasmid # 104448), and pEGFPC1-hVAP-B KD/MD (Addgene plasmid # 104450). ..

Article Title: STARD3 regulates lysosome positioning and contacts via a GSK3-controlled phosphorylation switch
Article Snippet: The GFP-MOSPD2 (WT Addgene plasmid # 186467; http://n2t.net/addgene:186467 ; RRID:Addgene_186467 and RD/LD mutant Addgene plasmid # 186468; http://n2t.net/addgene:186468 ; RRID:Addgene_186468), pQCXIP mScarlet-ER [TM(SAC1)] (Addgene plasmid # 186572; http://n2t.net/addgene:186572 ; RRID:Addgene_186572), and Flag-tagged STARD3 expression vectors were previously described (Alpy et al, ; Di Mattia et al, ; Zouiouich et al, ). .. The retroviral expression vector pQCXIP encoding GFP-VAP-A (WT and KD/MD mutant) and GFP-VAP-B (WT and KD/MD mutant) were constructed by cloning PCR-amplified fragments from pEGFPC1-hVAP-A (Addgene plasmid # 104447), pEGFPC1-hVAP-A KD/MD (Addgene plasmid # 104449), pEGFPC1-hVAP-B (Addgene plasmid # 104448), and pEGFPC1-hVAP-B KD/MD (Addgene plasmid # 104450). ..

Expressing:

Mutagenesis:

Construct:

Cloning:

Polymerase Chain Reaction:

Plasmid Preparation:

Article Title: The structure and global distribution of the endoplasmic reticulum network are actively regulated by lysosomes
Article Snippet: A pair of flat-ended platinum electrodes (Sigma-Aldrich) is held in place by a manual micromanipulator (World Precision Instruments) at the ends of the transverse channel. .. A glass capillary with a fine tip containing plasmid solution is inserted into the eye primordium of stage 26–30 embryos to inject 8 × 5- to 8-nl doses of pEGFPC1-hVAP-A (1 μg/μl; Addgene #104447) or pEGFPC1-hVAP-A KD/MD (Addgene #104449) plasmid driven by an air-pressured injector, such as a Picospritzer (Parker Hannifin). .. Immediately following the plasmid injection, eight electric pulses of 50-ms duration at 1000-ms intervals are delivered at 18 V by a square wave generator, such as the TSS20 Ovodyne electroporator (Intracel).

Article Title: The structure and global distribution of the endoplasmic reticulum network is actively regulated by lysosomes
Article Snippet: A pair of flat-ended platinum electrodes (Sigma) is held in place by a manual micromanipulator (World Precision Instruments) at the ends of the transverse channel. .. A glass capillary with a fine tip containing plasmid solution is inserted into the eye primordium of stage 26-30 embryos to inject 8−5-8 nl doses of 1 μg/μl of pEGFPC1-hVAP-A (Addgene #104447) or pEGFPC1-hVAP-A KD/MD (Addgene #104449) plasmid driven by an air-pressured injector, such as a Picospritzer (Parker Hannifin). .. Immediately following the plasmid injection, 8 electric pulses of 50 ms duration at 1000 ms intervals are delivered at 18 V by a square wave generator, such as the TSS20 OVODYNE electroporator (Intracel).

Article Title: The structure and global distribution of the endoplasmic reticulum network are actively regulated by lysosomes
Article Snippet: pEGFPC1-hVAP-A , Gift from C. Tomasetto , Addgene plasmid #104447. .. pEGFPC1-hVAP-A KD/MD , Gift from M. Davidson , Addgene plasmid #104449. .. mEmerald-Rab5a-7 , Gift from M. Davidson , Addgene plasmid #54243.

Review