Journal: bioRxiv

Article Title: Human breast milk extracellular vesicles from donors with asthma differentially the modulate release of inflammatory cytokines by primary human airway smooth muscle cells in a recipient-cell specific manner

doi: 10.64898/2026.03.02.709065

Figure Lengend Snippet: (A) Schematic representation of BM-EVs isolation and characterization. BM was collected from 3-4 months post-partum mothers with and without asthma diagnosis. BM-EVs were isolated by SEC (qEV original columns, Izon Science Ltd) and analyzed for size, concentration and zeta potential through TRPS using the qNano Gold instrument (Izon Science Ltd.). BM-EVs were concentrated for protein yield quantification through MicroBCA Protein Assay kit (Thermo Scientific), Western blot analysis for exosome and microvesicles markers and hASM cells treatment for cytokines release analysis. (B) TEM showing exosomal morphology and approximate size of BM-EVs (scale bar 100[nm). (C) Western blotting was performed (12% SDS-PAGE) and Ponceau S staining used as a loading control. Proteins enriched in exosomes (CD9, CD81, CD63, TSG101, HSP70 and Flotillin-1), in microvesicles (MMP2 and ARF6), and lipoproteins (APO-A1) were analysed. F7 to 9 were considered exosome-rich while microvesicles and lipoprotein-poor. CL: cell lysate from breast tissue, M: marker lane.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-TSG101 (T5701, Sigma-Aldrich Co, 1:200), rabbit polyclonal anti-CD63 (SAB4301607, Sigma-Aldrich Co, 1:200), mouse monoclonal anti-CD81 (sc-166029, Santa Cruz Biotechnology, 1:200), mouse monoclonal anti-CD9 (CBL162, Sigma-Aldrich Co, 1:100), rabbit polyclonal anti-Flotillin-1 (F1180, Sigma-Aldrich Co, 1:200), mouse monoclonal anti-heat shock protein 70 (HSP70) (H5147, Sigma-Aldrich Co, 1:500), mouse monoclonal anti-ARF6 (sc-7971, Santa Cruz Biotechnology, 1:200), mouse monoclonal anti-MMP2 (sc-13595, Santa Cruz Biotechnology, 1:200) and mouse monoclonal anti-Apolipoprotein A1 (Apo-A1) (0650-0050, Bio-Rad Laboratories, 1:200).

Techniques: Isolation, Biomarker Discovery, Concentration Assay, Zeta Potential Analyzer, Western Blot, SDS Page, Staining, Control, Marker

Journal: bioRxiv

Article Title: Human breast milk extracellular vesicles from donors with asthma differentially the modulate release of inflammatory cytokines by primary human airway smooth muscle cells in a recipient-cell specific manner

doi: 10.64898/2026.03.02.709065

Figure Lengend Snippet: (A) Equal amounts of protein (5 µg/mL) of BM-EVs from mothers with and without asthma were subjected to SDS-PAGE (12% SDS) and the expression of proteins enriched in exosomes [CD81 (22 kDa), CD63 (28 kDa), CD9 (25 kDa), Flotillin-1 (48 kDa), TSG101 (46 kDa), HSP70 (70 kDa)], in microvesicles [MMP2 (63kDa)], and in lipoproteins [APO-A1 (25kDa)] were quantified. Ponceau or Coomassie staining was used as a loading control to normalize protein content for all markers. (B) CD81 levels remained unchanged between the groups, (C) while asthma BM-EVs expressed ∼86% lower CD63 (p=0.0224) and (D) ∼ 24% lower CD9 (p=0.0646). (E) Flotillin-1 expression was lower by ∼40% in asthma BM-EVs compared to control (p=0.0196). (F) TSG101 levels remained unchanged, (G) while HSP70 was ∼69% lower in the asthma BM-EV (p=0.08). (H) APO-A1 was used as a purity control to investigate the presence of lipoproteins in the samples and did not show difference between groups. BM-EVs did not show any expression for MMP2 protein. Data were analyzed using an unpaired Student’s t-test with p<0.05 considered as significant and expressed as mean ± standard error (N=5).

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-TSG101 (T5701, Sigma-Aldrich Co, 1:200), rabbit polyclonal anti-CD63 (SAB4301607, Sigma-Aldrich Co, 1:200), mouse monoclonal anti-CD81 (sc-166029, Santa Cruz Biotechnology, 1:200), mouse monoclonal anti-CD9 (CBL162, Sigma-Aldrich Co, 1:100), rabbit polyclonal anti-Flotillin-1 (F1180, Sigma-Aldrich Co, 1:200), mouse monoclonal anti-heat shock protein 70 (HSP70) (H5147, Sigma-Aldrich Co, 1:500), mouse monoclonal anti-ARF6 (sc-7971, Santa Cruz Biotechnology, 1:200), mouse monoclonal anti-MMP2 (sc-13595, Santa Cruz Biotechnology, 1:200) and mouse monoclonal anti-Apolipoprotein A1 (Apo-A1) (0650-0050, Bio-Rad Laboratories, 1:200).

Techniques: SDS Page, Expressing, Staining, Control

Journal: Journal of Extracellular Biology

Article Title: Effect of a 12‐Week Endurance Training Program on Circulating Extracellular Vesicle Proteome in Sedentary Adults With Obesity

doi: 10.1002/jex2.70087

Figure Lengend Snippet: Extracellular vesicle isolation method from plasma samples. (A) Differential centrifugations followed by filtration and size exclusion chromatography (SEC). (B) Protein concentration (line) on the 14 SEC fractions, combined with western blot analysis using EV‐markers (ALIX and CD9) and Apolipoprotein A1 (APO A1) and B (APO B) as non‐EV markers on fractions 7 to 14. Fractions 7 to 9 are considered EV‐rich and protein‐ and lipoprotein‐poor and were pooled. (C) Transmission electron microscopy image of the pooled fractions (negative staining), scale bar refers to 1 µm and 200 nm. (D) Particles size distribution in pooled fractions analysed by NTA.

Article Snippet: The following antibodies were applied: Apolipoprotein (APO) B (1:1000, Santa Cruz, sc‐376818), APO A1 (1:1000, Santa Cruz, sc‐393636), ALG‐2‐interacting protein X (ALIX) (1:500, Cell Signaling Technology (CST), E6P9B), cluster of differentiation (CD) 9 (1:1000, CST, D3H4P), CD63 (1:1000, Santa Cruz, sc‐5275), CD81 (1:1000, CST, D3N2D), tumour susceptibility gene 101 (TSG101) (1:1000, Abcam, Ab30871), calnexin (1:1000, CST, 2433), LC3b (1:1000, Sigma, SAB4200361), TOM20 (1:1000, CST, 42406), peroxisome proliferator‐activated receptor gamma, coactivator 1 alpha (PGC‐1α) (1:1000, Santa Cruz, sc517380), oxidative phosphorylation (OXPHOS) (1:1000, Abcam, Ab110413 ), vascular endothelial growth factor A (VEGF‐A) (1:500, Santa Cruz, sc‐7269), citrate synthase (CS) (1:1000, CST, D7V8B), succinate dehydrogenase A (SDHA) (1:1000, Santa Cruz, sc‐390381), extracellular signal‐regulated kinase (ERK) 1/2 (1:1000, CST, L34F12), p‐ERK 1/2 (1:1000, CST, D13.14.4E), NFκB (1:1000, CST, D14E12), p‐NFκB (1:1000, CST, 93H1), hypoxia inducible factor 1 alpha (HIF‐1α) (1:500, CST, D2U3T), peroxiredoxin (PRDX) 1 (1:1000, CST, D5G12), actin (1:5000, BD Biosciences, 612656) and eukaryotic translation elongation factor 2 (eEF2) (1:1000, CST, 2332).

Techniques: Isolation, Clinical Proteomics, Filtration, Size-exclusion Chromatography, Protein Concentration, Western Blot, Transmission Assay, Electron Microscopy, Negative Staining