unconjugated mouse anti human cd81  (Novus Biologicals)

Journal: bioRxiv

Article Title: Extracellular vesicles stimulate smooth muscle cell migration by presenting collagen VI

doi: 10.1101/2023.08.17.551257

Figure Lengend Snippet: A , Proteomic profiling of pairwise collected carotid atherosclerotic plaques and adjacent intact arterial segments. Venn diagram shows the number of plaque-specific (n = 213) and intact-specific (n = 111) proteins as well as the number of proteins which are common for both vascular regions (1,368). B . Partial least-squares discriminant analysis indicates clear classification pattern between the carotid plaques (indicated by red triangles) and adjacent intact arterial segments (indicated by blue circles). N = 14. C . Volcano plot illustrates that 46 proteins are significantly overexpressed in plaques whilst 13 proteins are significantly upregulated in adjacent intact arterial segments. D , Manders’ split colocalization coefficient for the overlap of FN with CD81 (M1) and CD81 with FN (M2). Neointima region as in . E , Atherosclerotic plaques were co-stained for fibronectin (FN) and sEV marker, CD81. Cell nuclei were counterstained with DAPI. Main figure: x200 magnification, size bar, 50 µm. Box: x400 magnification, size bar, 15µm. Note an accumulation of FN in the neointima. F, Spatial distribution of FN and CD81 in the neointima. Note high overlap between FN and CD81 in the extracellular matrix. x200 magnification, size bar, 50 µm. G, Quantification of FN content in atherosclerotic plaques. Samples were analysed by western blot and bands intensity was quantified in ImageJ. Fold change was calculated as ratio of band intensity in the atherosclerotic plaque to band intensity in the adjacent intact arterial segments normalised to GAPDH. Note that FN content is elevated in atherosclerotic plaques relative to the adjacent intact arterial segments. Paired t-test. H , FN is presented on the surface of the VSMC-derived sEV along with α5β1 integrin. VSMC sEVs were immobilised on the 4µm beads. sEV-beads and VSMCs were stained with the antibodies (filled graphs) in non-permeabilised conditions and analysed by flow cytometry.

Article Snippet: Upon the sectioning, vascular tissues were dried at room temperature for 30min, fixed and permeabilised in ice-cold acetone for 10min, incubated in 1% bovine serum albumin (Cat. No. A2153, Sigma-Aldrich) for 1h to block non-specific protein binding, stained with unconjugated mouse anti-human CD81 (M38 clone, NBP1-44861, 1:100, Novus Biologicals) and rabbit anti-human fibronectin (F14 clone, ab45688, 1:250, Abcam) primary antibodies and incubated at 4°С for 16h.

Techniques: Staining, Marker, Western Blot, Derivative Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Extracellular vesicles stimulate smooth muscle cell migration by presenting collagen VI

doi: 10.1101/2023.08.17.551257

Figure Lengend Snippet: A, Proteomic analysis of VSMC-derived sEVs and EV. Venn diagram. N=3. B, Protein enrichment in the EV and sEV proteome. Heat Map. N=3. C, Western blot validation of sEV cargos. EV and sEV were isolated from VSMC’s conditioned media by differential ultracentrifugation and analysed by western blotting. Representative image from N=3. D, VSMC adhesion is regulated by collagen VI loaded to sEV. FN matrices were incubated with sEV and anti-collagen VI antibody (COLVI IgG) or control IgG. Cell adhesion was tracked by using ACEA’s xCELLigence Real-Time Cell Analysis. ANOVA, N=3. E, F, J, sEV promote directional VSMC invasion. VSMCs were treated with control siRNA (Scramble) or collagen VI-specific siRNA pools for 24h and were seeded to the FN-enriched Matrigel matrix in μ-Slide Chemotaxis assay and stained with Draq5. Cell tracking was conducted by OperaPhenix microscope for 12h and cell invasion parameters were quantified using Columbus. Kolmogorov-Smirnov test, *, p<0.05 I , Real-time PCR analysis of expression of CD9, CD63, CD81, COL6A3, EDIL3 and TGFBI in atherosclerotic plaque. *, p<0.05, Paired t-test, N=5.

Article Snippet: Upon the sectioning, vascular tissues were dried at room temperature for 30min, fixed and permeabilised in ice-cold acetone for 10min, incubated in 1% bovine serum albumin (Cat. No. A2153, Sigma-Aldrich) for 1h to block non-specific protein binding, stained with unconjugated mouse anti-human CD81 (M38 clone, NBP1-44861, 1:100, Novus Biologicals) and rabbit anti-human fibronectin (F14 clone, ab45688, 1:250, Abcam) primary antibodies and incubated at 4°С for 16h.

Techniques: Derivative Assay, Protein Enrichment, Western Blot, Biomarker Discovery, Isolation, Incubation, Control, Cell Analysis, Chemotaxis Assay, Staining, Cell Tracking Assay, Microscopy, Real-time Polymerase Chain Reaction, Expressing