Journal: Developmental cell

Article Title: Exosomal microRNA transport from salivary mesenchyme regulates epithelial progenitor expansion during organogenesis.

doi: 10.1016/j.devcel.2016.12.001

Figure Lengend Snippet: (a) Exosomes (Exo) isolated by sequential centrifugation were analyzed by transmission electron microscopy (TEM, Scale bar = 50 nm) and Western blot for the exosome markers Alix and TSG101. Intact SMG and NIH3T3 cell lysates are positive controls. (b) Bioanalyzer analysis shows that small RNAs in exosomes were resistant to RNase A degradation. Arrows indicate the peak of small RNA before and after RNase A treatment. FU; fluorescence units. N = 3, graph is a representative experiment.

Article Snippet: To confirm that ceramide-labeled vesicles released by the mesenchyme contained exosomes, we co-stained the mesenchyme sitting on top of the ECM with antibodies to TSG101, an exosome marker ( Fig. S2 ).

Techniques: Isolation, Centrifugation, Transmission Assay, Electron Microscopy, Western Blot, Fluorescence

Journal: Oncotarget

Article Title: Identification of prostate cancer biomarkers in urinary exosomes

doi:

Figure Lengend Snippet: A. Removal of uromodulin after 2000 x g centrifugation. Urine samples (30 μl in sample buffer 4x) before (start urine) and after 2000 x g centrifugation (2000g, sup.) were loaded onto the gel. The pellet obtained after 2000 x g centrifugation was solubilized in a similar volume of water as the volume of the supernatant, and 30 μl in sample buffer 4x were loaded onto the gel (2000g, pellet). Asterisks in Coomassie stained gels (upper part) indicate uromodulin as confirmed by Western blot (lower part). Sup: Supernatant. Std: molecular weight (kD) standard. B. Urinary exosomes were isolated by ultracentrifugation and 10 μg were run in a 4-20% SDS-PAGE. Exosomal proteins were Coomassie stained. Std: molecular weight (kD) standard. C. Urinary exosomes labeled with mouse anti-CD63 followed by rabbit-anti-mouse, and then by 10 nm Protein A-gold conjugates were inspected by electron microscopy. D. Identification of CD9, CD81 and Tsg101 in urinary exosomes (2 μg) by Western blot. E . Five exosome markers detected by MS, quantified by Top 3 TIC ( n = 15; ± SEM).

Article Snippet: The antibodies used for Western blotting were: mouse anti-Tsg101 (BD Biosciences, Heidelberg, Germany); rabbit anti-CD9 (Abcam, Cambridge, UK); mouse anti-CD81 (Ancell Corporation, Bayport, MN, USA), rabbit anti-uromodulin (St. Cruz Biotechnology Inc., Dallas, TX, USA).

Techniques: Centrifugation, Staining, Western Blot, Molecular Weight, Isolation, SDS Page, Labeling, Electron Microscopy

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) Protein expression of precipitated EVs. The serum collected from seven mice of wild-type (WT) and EML4-ALK transgenic mouse (TL group) was subjected to Western blot analysis with anti-CHL1, anti-α2 integrin, anti-β1 integrin, and anti-FGFR3 antibodies, which were cancer cell membrane BiCAT molecules previously reported. ( B ) Western blot analysis for TSG101 antigen detection in Sephacryl S-500 fractions. The fractions were concentrated with Nanosep ® centrifugal unit, and then subjected to Western blot analysis with anti-TSG101 antibody. TSG101-(Ub)n indicates ubiquitinated TSG101.

Article Snippet: After blocking with 5% skim milk solution, transferred membrane was reacted with first antibodies as follows: anti-mouse and human CHL1 antibody (AF2147 and MAB2126; R&D systems, MN; 1 μg/ml), HRP-conjugated anti-mouse and human CHL1 antibody (described above), anti-α2 integrin antibody (ab133557; Abcam; 1 μg/ml), anti-β1 integrin antibody (610467; BD transduction laboratories, NJ; 0.25 μg/ml), anti-FGFR3 antibody (c-15; Santa cruz, TX; 0.2 μg/ml 5% BSA-PBS), anti-TSG101 antibody (EXOAB-TSG101-1; System Biosciences, CA; 1:500), anti-CD63 antibody (EXOAB-CD63A-1; System Biosciences, CA; 1:500), anti-CD5L antibody (AF2834; R&D systems, MN; 0.4 μg/ml), anti-Pregnancy Zone Protein (PZP) antibody (PAG324Ra01; CLOUD-CLONE, TX; 0.5 μg/ml), anti-SLC4A1 antibody (18566-1-AP; PROTEINTECH, IL; 0.6 μg/ml), anti-Thrombospondin 1 (THBS1) antibody (PAA611Hu01; CLOUD-CLONE, TX; 0.5 μg/ml), and anti-caspase 14 antibody (MAB8215; R&D systems, MN; 0.5 μg/ml).

Techniques: Expressing, Transgenic Assay, Western Blot

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A, B ) Western blot analysis of TSG101 ( A ) and CD63 ( B ) in serum EVs from WT, TL, and TS using anti-TSG101 and CD63 antibody. Arrows indicate the detected band of monomer TSG101 and CD63. The black bar indicates wide range of molecular weight due to an ubiquitination in TSG101 (TSG101-(Ub)n). ( C ) Western blot analysis of EMARS products with anti-CHL1 antibody. The EMARS products were concentrated and purified by immunoprecipitation with the anti-fluorescein antibody Sepharose. The resulting samples were subjected to SDS-PAGE analysis with fluorescence detection. “IP” indicates the immunoprecipitated samples, and “Lys” indicates the lysate samples before immunoprecipitation. Arrow indicates the detected band of CHL1. Asterisk indicates unknown bands (predicted as non-specific or partial fragments).

Article Snippet: After blocking with 5% skim milk solution, transferred membrane was reacted with first antibodies as follows: anti-mouse and human CHL1 antibody (AF2147 and MAB2126; R&D systems, MN; 1 μg/ml), HRP-conjugated anti-mouse and human CHL1 antibody (described above), anti-α2 integrin antibody (ab133557; Abcam; 1 μg/ml), anti-β1 integrin antibody (610467; BD transduction laboratories, NJ; 0.25 μg/ml), anti-FGFR3 antibody (c-15; Santa cruz, TX; 0.2 μg/ml 5% BSA-PBS), anti-TSG101 antibody (EXOAB-TSG101-1; System Biosciences, CA; 1:500), anti-CD63 antibody (EXOAB-CD63A-1; System Biosciences, CA; 1:500), anti-CD5L antibody (AF2834; R&D systems, MN; 0.4 μg/ml), anti-Pregnancy Zone Protein (PZP) antibody (PAG324Ra01; CLOUD-CLONE, TX; 0.5 μg/ml), anti-SLC4A1 antibody (18566-1-AP; PROTEINTECH, IL; 0.6 μg/ml), anti-Thrombospondin 1 (THBS1) antibody (PAA611Hu01; CLOUD-CLONE, TX; 0.5 μg/ml), and anti-caspase 14 antibody (MAB8215; R&D systems, MN; 0.5 μg/ml).

Techniques: Western Blot, Molecular Weight, Purification, Immunoprecipitation, SDS Page, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Salidroside Inhibits CCl 4 -Induced Liver Fibrosis in Mice by Reducing Activation and Migration of HSC Induced by Liver Sinusoidal Endothelial Cell-Derived Exosomal SphK1

doi: 10.3389/fphar.2021.677810

Figure Lengend Snippet: Characteristics of serum exosomes and their effect on migration of HSC. (A) : Serum exosomes had a double-layer membrane structure. (B) : Expression of TSG101 and CD9 in serum exosomes from human and mice. (C) : Expression of SphK1 in serum exosomes from human ( n = 6, * p < 0.05, vs. Normal-exo.) (D) : Effects of serum exosomes from different populations on LX-2 migration ( n = 6, * p < 0.05, vs Normal-exo.) (E) : Expression of SphK1 in serum exosomes from different groups of mice ( n = 15, ** p < 0.01, vs. Cr-exo, # p < 0.05, vs. M-exo,) (F) : Effects of serum exosomes from different groups of mice on JS 1 migration ( n = 15, ** p < 0.01, vs Cr-exo, ## p < 0.01, vs. M-exo.) TSG101: tumor susceptibility gene 101.

Article Snippet: SphK1, SphK2, S1PR1, S1PR2, S1PR3, CD9, and tumor susceptibility gene 101 (TSG101) antibodies were procured from ImmunoWay Biotechnology Company (Suzhou, China).

Techniques: Migration, Membrane, Expressing

Journal: Frontiers in Pharmacology

Article Title: Salidroside Inhibits CCl 4 -Induced Liver Fibrosis in Mice by Reducing Activation and Migration of HSC Induced by Liver Sinusoidal Endothelial Cell-Derived Exosomal SphK1

doi: 10.3389/fphar.2021.677810

Figure Lengend Snippet: Effects of Sal on exosomal SphK1-induced LX-2 migration and activation. (A) : Expression of SphK1 in transfected cells was detected by WB. (B) : Expression of SphK1 in transfected cells was detected by PCR. (C) : Expression of TSG101 and CD9 in exosomes from culture mediums. (D,E) : Expression of SphK1 and FN in exosomes from culture mediums of transfected cells. ( n = 3, ** p < 0.01, vs Cr-exo.) (F) : Sal inhibited exosomal SphK1-induced LX-2 migration and activation. (G) : Sal inhibited exosomal SphK1-induced AKT phosphorylation in LX-2. ( n = 3, * p < 0.05, ** p < 0.01, vs. Cr-exo; # p < 0.05, ## p < 0.01, vs. SphK1 + -exo.).

Article Snippet: SphK1, SphK2, S1PR1, S1PR2, S1PR3, CD9, and tumor susceptibility gene 101 (TSG101) antibodies were procured from ImmunoWay Biotechnology Company (Suzhou, China).

Techniques: Migration, Activation Assay, Expressing, Transfection, Phospho-proteomics