flotillin1  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Microfluidic Extraction of Extracellular Vesicle-Associated Nucleic Acids for Downstream Molecular Analysis

doi: 10.64898/2026.10.12.758106

Figure Lengend Snippet: Preparation and characterization of a standardized EV model sample. (A) Preparation of EV- spike from Hec-1-A cell conditioned medium (B) Isolation of EVs from biological matrix. Characterization of EV-spike by (C) transmission electronic microscopy (TEM), (D) Western-blotting for the EV-associated proteins ALIX, CD63, flotillin-1, and the cellular contamination marker calnexin (E) nanoparticle tracking analysis (NTA). (F) Formulating models samples by introducing EV-spike into sample matrix (plasma or serum), (G) Characteristics of model samples and their components for particle concentration, size, protein and nucleic acid content.

Article Snippet: Following electrophoresis, proteins were transferred onto PVDF membranes by wet transfer at 100 V for 90 min. Membranes were subsequently blocked with 5% non-fat milk prepared in PBS-T for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies against CD63 (1:500, Santa Cruz), flotillin (1:500, Proteintech), Alix (1:500, Thermo Fisher Scientific) and calnexin (1:5000, Abcam).

Techniques: Isolation, Transmission Assay, Microscopy, Western Blot, Marker, Analysis, Clinical Proteomics, Concentration Assay, Transmission Electron Microscopy, Analyte Concentration

Journal: Signal Transduction and Targeted Therapy

Article Title: Breast cancer-associated fibroblast-derived exosomal miR-1290 promotes triple-negative breast cancer cell proliferation

doi: 10.1038/s41392-026-02922-y

Figure Lengend Snippet: miR-1290 is upregulated in breast cancer and BCAF/MB-231-derived exosomes. a Nanoparticle tracking analysis of exosomes isolated from BCAFs and MB-231 cells showing their size distribution. The mean size and standard deviation (SD) are indicated for each sample. b TEM images of exosomes isolated from BCAFs (upper) and MB-231 cells (lower) showing the characteristic morphology of the exosomes. Scale bar, 100 nm. c Western blot analysis of exosome markers (CD63, CD81, and flotillin-1), CAF markers (α-SMA and FAP) and cellular markers (GM130 and H3) in BCAF cells, MB-231 cells and their derived exosomes. d BCAF-derived exosome uptake by TNBC cells. PKH67-labeled BCAF-derived exosomes (green) were incubated with BT-549 and Hs578T cells for 1 hour. The cell nuclei were stained with Hoechst 33342 (blue). Scale bar, 20 μm. e Heatmap showing miRNA expression profiles in exosomes derived from BCAFs and MB-231 cells. Red indicates high expression, and blue indicates low expression. miR-1290 (highlighted in red) is highly expressed in both exosome types. f Volcano plots showing differentially expressed miRNAs between breast cancer or TNBC samples and normal controls in five independent GEO datasets ( GSE37963 , GSE45666 , GSE45498 , GSE38167 , and GSE61438 ). Red and blue dots represent upregulated and downregulated miRNAs, respectively, selected using fold-change and nominal p value cutoffs (|log2(fold change)| > 0.58 and nominal p < 0.05, two-tailed Welch’s t test). g Venn diagram analysis of upregulated miRNAs across the five GEO datasets revealed four commonly upregulated miRNAs. h Violin plot showing miR-1290 expression levels in serum samples from normal individuals ( N , n = 2686) and breast cancer patients ( P , n = 1280) from the GSE73002 dataset. The expression of miR-1290 was significantly upregulated in the breast cancer patient samples (*** p < 0.001, Mann‒Whitney U test). i Analysis of serum exosomal miR-1290 levels in normal ( N , n = 20) and TNBC patient ( P , n = 39) samples collected at Keimyung University Dongsan Hospital. The results support the potential of miR-1290 as a candidate diagnostic marker in an independent cohort (*** p < 0.001, Mann‒Whitney U test). j Kaplan‒Meier plot of overall survival for TNBC patients based on serum exosomal miR-1290 expression levels from Keimyung University Dongsan Hospital ( p = 0.006, log-rank test)

Article Snippet: Membranes were incubated with the following primary antibodies: CD63 (1:200; ABclonal, A5271), CD81 (1:200; ABclonal, A5270), flotillin-1 (1:1000; ABclonal, A6220), α-SMA (1:1000; ABclonal, A17910), FAP (1:1000; ABclonal, A23789 ), GM130 (1:1000; Abcam, ab52649), H3 (1:1000; Cell Signaling Technology, 4620S), ACTB (1:20,000; ABclonal, AC026), TBC1D4 (1:200; Cell Signaling Technology, 2670S), P70S6K (1:500; Cell Signaling Technology, 9202S), phospho-P70S6K (1:500; ABclonal, AP0564), and Ki67 (1:1000; Abcam, ab16667).

Techniques: Derivative Assay, Exosomes, Analysis, Isolation, Standard Deviation, Morphology, Western Blot, Labeling, Incubation, Staining, Expressing, Two Tailed Test, Blood/Plasma/Serum Samples, Diagnostic Assay, Marker

Journal: Journal of Extracellular Biology

Article Title: Dysregulation of Extracellular Vesicle Concentration, MicroRNAs, and Surface Proteins in Patients With Niemann‐Pick Disease Type C

doi: 10.1002/jex2.70136

Figure Lengend Snippet: SEC enriches for EVs from fibroblast conditioned media . ( A ) EV enrichment method for fibroblast conditioned media. ( B, C ) Immunoblots of individual ( B ) or pooled ( C ) SEC fractions from CTL fibroblast media show enrichment for EV proteins Flotillin‐1, CD63, and CD81, without detectable levels of other proteins (total protein stain) or cell lysate protein GM130. ( D ) MRPS histogram shows the distribution of particles in pooled EV fractions isolated from CTL fibroblast media. Mean particle count, size, and coefficient of variation are listed.

Article Snippet: Primary antibodies included NPC1 (EPR5209) 1:1000 (134113, Abcam); CD63 (H5C6) 1:5,000 (556019, BD Biosciences); CD81 (B‐11) 1:100 (sc‐166029, Santa Cruz Biotechnology); Flotillin‐1 1:10,000 (ab133497, Abcam); GM130 1:1000 (53420SS, Novus Biologicals); and Lamp1 (D2D11) 1:1000 (9091S XP, Cell Signaling Technology).

Techniques: Western Blot, Staining, Isolation

Journal: Journal of Extracellular Biology

Article Title: Dysregulation of Extracellular Vesicle Concentration, MicroRNAs, and Surface Proteins in Patients With Niemann‐Pick Disease Type C

doi: 10.1002/jex2.70136

Figure Lengend Snippet: SEC enriches for EVs from human CSF . ( A ) EV enrichment method for CSF. ( B, C ) Immunoblots of individual ( B ) or pooled ( C ) SEC fractions from CTL CSF show enrichment for EV proteins Flotillin‐1 and CD81 without detectable levels of bulk CSF proteins (total protein stain, albumin, ApoA1). ( D ) MRPS histogram shows the distribution of particles in pooled EV fractions from CTL CSF. Mean particle count, size, and coefficient of variation are listed. ( E ) TEM of representative NPC CSF EV sample. Black arrows indicate particles of ∼100 nm; white arrows indicate particles of ∼50 nm.

Article Snippet: Primary antibodies included NPC1 (EPR5209) 1:1000 (134113, Abcam); CD63 (H5C6) 1:5,000 (556019, BD Biosciences); CD81 (B‐11) 1:100 (sc‐166029, Santa Cruz Biotechnology); Flotillin‐1 1:10,000 (ab133497, Abcam); GM130 1:1000 (53420SS, Novus Biologicals); and Lamp1 (D2D11) 1:1000 (9091S XP, Cell Signaling Technology).

Techniques: Western Blot, Staining