Journal: International Journal of Molecular Sciences

Article Title: Proteomic Profiling of Two Distinct Populations of Extracellular Vesicles Isolated from Human Seminal Plasma

doi: 10.3390/ijms21217957

Figure Lengend Snippet: Isolation and characterization of LEV and SEV. Total EV in seminal plasma of vasectomized men were collected by UC at the interface of a sucrose block gradient. LEV and SEV were separated by their distinct velocities during upward displacement into a continuous sucrose density gradient. ( A ) Gradient fractions were analyzed by SDS-PAGE followed by Sypro ruby staining for total protein. ( B ) Gradient fractions were analyzed by immunoblotting for the presence of EV associated proteins, including CD9, CD81, PSCA, Galectin-3, CD63, HSP70, Annexin A1, and GLIPR2. Molecular weight markers are indicated on the left in kDa. ( C ) Particles in the LEV and SEV containing fractions were analyzed by TEM. Scale bar, 500 nm. Arrows exemplify incidental SEV in the LEV isolate. ( D ) Size distribution of the particles in LEV and SEV isolates as determined by NTA.

Article Snippet: The primary antibodies used were mouse anti human CD9 (HI9a, 312102, Biolegend, San Diego, California, USA, 1:2000); mouse anti human CD81 (B11, sc-166029, Santa Cruz Biotechnology, Dallas, Texas, USA, 1:500); mouse anti human HSP70 (N27F3-4, ADI-SPA-820-D, Enzo, Bruxelles, Belgium, 1:1000); mouse anti human Flotillin-1 (clone 18, 610821, BD Biosciences, San Jose, California, USA, 1:1000); mouse anti-human PSCA (clone 7F5, sc-80654, Santa Cruz Biotechnology, Dallas, Texas, USA, 1:1000); mouse anti human Annexin A1 (clone 29, 610066, BD Biosciences, San Jose, California, USA, 1:1000); mouse anti human CD47 (B6H12, sc-12730, Santa Cruz Biotechnology, Dallas, Texas, USA, 1:500); rat anti human galectin-3 (M3/38, CL8942B, CEDARLANE, Burlington, Vermont, USA, 1:500) and rabbit anti human GLIPR2/GAPR-1 (Eberle et al., 2002, 1:5000, kindly provided by J.B. Helms).

Techniques: Isolation, Clinical Proteomics, Blocking Assay, SDS Page, Staining, Western Blot, Molecular Weight

Journal: Biology of reproduction

Article Title: Identification of distinct populations of prostasomes that differentially express prostate stem cell antigen, annexin A1, and GLIPR2 in humans.

doi: 10.1095/biolreprod.111.095760

Figure Lengend Snippet: FIG. 1. Fractionation by column chromatography. Membrane vesicles were collected from seminal plasma by ultracentrifugation and then separated by Sephacryl S-1000 column chromatography. A) CD9 and PSCA immunoblots of protein-containing fractions. Molecular weight markers are indicated in Mr 3 103. B and C) Representative whole mount transmission electron micrographs of membrane vesicles from pooled void volume fractions (28–34 from A, presented in B) and retained membrane vesicles (pooled fractions 44–54 from A, presented in C). Bars ¼ 500 nm. The data shown are representative of three independent experiments.

Article Snippet: Other blots were probed with mouse anti-human PSCA (clone 7F5; 1:1000; Santa Cruz Biotechnology Inc.), rabbit anti-human PSCA (1:250; Acris Antibodies Inc.), rabbit anti-human GLIPR2 [29] (1:5000), or mouse anti-human annexin A1 (clone 29; 1:250; BD Biosciences) [30].

Techniques: Fractionation, Column Chromatography, Membrane, Clinical Proteomics, Western Blot, Molecular Weight, Transmission Assay

Journal: Biology of reproduction

Article Title: Identification of distinct populations of prostasomes that differentially express prostate stem cell antigen, annexin A1, and GLIPR2 in humans.

doi: 10.1095/biolreprod.111.095760

Figure Lengend Snippet: FIG. 2. Fractionation on sucrose density gradients. Membrane vesicles from the pooled column fractions 44–54 in Figure 1A were loaded either on top (A) or at the bottom (B) of sucrose gradients and centrifuged for 62 h. C) Membrane vesicles were loaded at the bottom of a linear sucrose gradient and centrifuged for 16 h. CD9 and PSCA in gradient fractions were detected by immunoblotting as indicated. Molecular weight markers are indicated (Mr3103) on the right of the image, and the densities of the fractions (g/ml) are shown below the blots. The data shown are representative of six independent experiments.

Article Snippet: Other blots were probed with mouse anti-human PSCA (clone 7F5; 1:1000; Santa Cruz Biotechnology Inc.), rabbit anti-human PSCA (1:250; Acris Antibodies Inc.), rabbit anti-human GLIPR2 [29] (1:5000), or mouse anti-human annexin A1 (clone 29; 1:250; BD Biosciences) [30].

Techniques: Fractionation, Membrane, Western Blot, Molecular Weight

Journal: Biology of reproduction

Article Title: Identification of distinct populations of prostasomes that differentially express prostate stem cell antigen, annexin A1, and GLIPR2 in humans.

doi: 10.1095/biolreprod.111.095760

Figure Lengend Snippet: FIG. 3. Membrane vesicles from high- and low-density fractions are distinct in size but both contain CD9 and PSCA. Two vesicle populations were separated by sucrose gradient fractionation as indicated in Figure 2C. Vesicles from high- and low-density fractions were immobilized, immunogold- labeled for CD9 or PSCA, and analyzed by whole mount transmission electron microscopy. Representative pictures from two independent experiments are shown. Labeled vesicles are indicated by arrows. Bars¼ 500 nm. Vesicle mean sizes and percentages of vesicles labeled for CD9 or PSCA are indicated in Table 1.

Article Snippet: Other blots were probed with mouse anti-human PSCA (clone 7F5; 1:1000; Santa Cruz Biotechnology Inc.), rabbit anti-human PSCA (1:250; Acris Antibodies Inc.), rabbit anti-human GLIPR2 [29] (1:5000), or mouse anti-human annexin A1 (clone 29; 1:250; BD Biosciences) [30].

Techniques: Membrane, Fractionation, Labeling, Transmission Assay, Electron Microscopy

Journal: Biology of reproduction

Article Title: Identification of distinct populations of prostasomes that differentially express prostate stem cell antigen, annexin A1, and GLIPR2 in humans.

doi: 10.1095/biolreprod.111.095760

Figure Lengend Snippet: FIG. 4. PSCA is absent on prostasomes from some individual donors. Prostasomes were collected from the seminal fluid of individual donors (1–12) and immunoblotted for CD9 (A). The samples were probed on parallel blots for PSCA using mouse anti-human PSCA (B) or rabbit anti- human PSCA (C). Molecular weight markers are indicated on the right (Mr 3 103).

Article Snippet: Other blots were probed with mouse anti-human PSCA (clone 7F5; 1:1000; Santa Cruz Biotechnology Inc.), rabbit anti-human PSCA (1:250; Acris Antibodies Inc.), rabbit anti-human GLIPR2 [29] (1:5000), or mouse anti-human annexin A1 (clone 29; 1:250; BD Biosciences) [30].

Techniques: Molecular Weight