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: SDS-PAGE and Western blot analysis were performed as previously described ( ) using the following antibodies: rabbit anti-GFP (1:2000; TP401, Torrey Pine Biolabs, RRID:AB_10013661), rabbit anti-MOSPD2 (1:1000; HPA003334, Sigma-Aldrich, RRID:AB_2146004), rabbit anti-mCherry (1:1000; ab167453, Abcam, RRID:AB_2571870), mouse anti-EEA1(1:1000; 610457, BD Biosciences, RRID:AB_397830), mouse anti-LAMP1 (1:1000; H4A3, DSHB, RRID:AB_2296838), mouse anti-LAMP2 (1:1000; H4B4, DSHB, RRID:AB_528129), mouse anti-VAP-A (1:1000; sc-293278, Santa Cruz Biotechnology, RRID:AB_2801294), anti-VAP-B [rabbit anti-VAP-B , rabbit anti-VAP-B (1:1000; HPA013144, Sigma-Aldrich, RRID:AB_1858717)], anti-STARD3 [rabbit polyclonal 605 , mouse monoclonal 3G11 ( )], rabbit anti-ORP1 (1:1000; ab131156, Abcam, RRID:AB_1155305), mouse anti-OSBP (1:1000; sc-365771, Santa Cruz Biotechnology, RRID:AB_10847232), rabbit anti-GAPDH (1:1000; G9545, Sigma-Aldrich, RRID:AB_796208) and mouse anti-actin (1:5000; ACT-2D7, Euromedex).
Techniques: Immunoprecipitation, Western Blot, Control, Incubation, Binding Assay, Staining, Recombinant, Mutagenesis, Purification, Expressing
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