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: For each MSP bait, 395 μL of streptavidin magnetic bead slurry (820 μL for the MBP control) (PureProteome Streptavidin Magnetic Beads, LSKMAGT10, Merck) in 1.5 mL tubes were washed twice with Holdup buffer.

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

Journal: Nature chemical biology

Article Title: A druggable redox switch on SHP1 controls macrophage inflammation

doi: 10.1038/s41589-026-02163-8

Figure Lengend Snippet: a,c , Intact protein MS of human recombinant wild type (WT) or Cys102 to Ser102 mutant (C102S) SHP1 (2 μM) incubated with DMSO, 5 or 10 molar equivalents of SCA1 for 24 h (4°C). b , Quantitative MS determination of the proportion of free cysteine in human recombinant SHP1 incubated with DMSO or 10 molar equivalents of SCA1 for 2 h (room temperature) ( n = 3). d , SHP1 binding by desthiobiotinylated SCA1 in iBMDM or THP-1 MDM lysates treated overnight (4°C) at the indicated concentrations and enriched by streptavidin pulldown. Immunoblots shown are representative of three independent experiments. e , SHP1 binding by SCA1 in iBMDM or THP-1 MDM cells treated at the indicated concentrations for 3 h (37°C) followed by competition for SHP1 binding with desthiobiotinylated SCA1 (10 μM). Densitometry analyses of SHP1 immunoblots from n = 3 independent experiments each are shown. Data are mean ± s.e.m. (in e ) or s.d. in ( b ). P values calculated using one-way ANOVA for multiple comparisons involving one independent variable or two-tailed Student’s t -tests for unpaired comparisons.

Article Snippet: 30 μl slurry streptavidin beads (Pierce high capacity streptavidin agarose, #20357) were added the next day, incubated for 3 h, 4°C, with rotation.

Techniques: Recombinant, Mutagenesis, Incubation, Binding Assay, Western Blot, Two Tailed Test