Journal: Scientific Reports

Article Title: Different populations of Wnt-containing vesicles are individually released from polarized epithelial cells

doi: 10.1038/srep35562

Figure Lengend Snippet: ( a ) Western blot analysis to examine relative amounts of exosome marker proteins, CD63, CD81, Tsg101, and Hsp70, recovered in P100 pellets of apical or basolateral medium of Wnt3a-expressing MDCK (MDCK-3a) cells. Equal amounts of P100 samples prepared from apical or basolateral medium were subjected to Western blotting. As standards, small amounts of cell lysates were also loaded. ( b ) The amounts of Wnt3a in S10 sup and P100 pellet from apical or basolateral medium of MDCK-3a cells, as well as the amount in the cell lysate, were analyzed by Western blotting. To compare the amounts of Wnt3a proteins between apical and basolateral media, an equal volume of S10 (10 μl each for apical or basolateral S10 pool) or equal amount of P100 (1/40 of P100 pellets prepared from 400 ml of apical or basolateral media) samples was loaded. In all MDCK cultures in this study, the same volume of medium was added to both apical and basolateral chambers. ( c–f ) Western blotting analysis for detection of activity and amount of Wnt proteins recovered in S10 sup, S100 sup or P100 pellet prepared from either the apical ( c ) or basolateral side ( e ) of MDCK cells. For monitoring of the Wnt3a activity, the amount of stabilized β-catenin induced by the addition of S10 sup, S100 sup or P100 pellet was quantified in L cells, in which β-catenin is undetectable without the addition of Wnt proteins (control; c,e ). In parallel, the amount of Wnt3a used in this activity assay was analyzed ( c,e ). The amounts of β-catenin and Wnt3a in S10, S100 and P100, shown in ( c ) or ( e ), were quantified using Image J software. The ratios of β-catenin to Wnt3a level are indicated in numerical values relative to that in S10, which was set as 100 ( d,f ). The results shown are the mean ± S.D. from 3 independent experiments. Full-length blots are presented in .

Article Snippet: The P100 pellet isolated from apical MDCK conditioned medium was incubated with 1 × 10 5 anti-CD63 or isotype control conjugated beads in 300 μL wash buffer (PBS containing 2% exosome-depleted FCS) overnight at 4 °C under gentle agitation.

Techniques: Western Blot, Marker, Expressing, Activity Assay, Software

Journal: Scientific Reports

Article Title: Different populations of Wnt-containing vesicles are individually released from polarized epithelial cells

doi: 10.1038/srep35562

Figure Lengend Snippet: ( a,b ) The P100 pellet of either apical ( a ) or basolateral ( b ) conditioned medium from Wnt3a-expressing MDCK (MDCK-3a) cells was subjected to 0.25–2 M continuous sucrose density-gradient centrifugation. Equal volumes of the collected fractions were analyzed by Western blotting to detect Wnt3a and several markers of exosomes, including Flotillin2 (Flo2), CD63, and Tsg101. In addition, contamination of the ER in the apical sample was examined by use of Calreticulin antibody. Distribution of Hsp70 in apical P100 fractionations is indicated separately in comparison with that of CD63. The results are representative of 4 independent experiments. ( c ) Immunoprecipitation analysis to detect Wnt3a in CD63-containing vesicles. The P100 pellet prepared from apically secreted conditioned medium was incubated with anti-CD63-beads or IgG isotype-beads, and the complexes on the beads were recovered and examined with anti- Wnt3a and CD63 antibodies. ( d ) Immuno-electron microscopy of Wnt3a-containing exosomes released into apical and basolateral media. Vesicles stained with anti-Wnt3a antibody and gold particle-conjugated second antibody were observed by transmission electron microscopy. Full-length blots are presented in .

Article Snippet: The P100 pellet isolated from apical MDCK conditioned medium was incubated with 1 × 10 5 anti-CD63 or isotype control conjugated beads in 300 μL wash buffer (PBS containing 2% exosome-depleted FCS) overnight at 4 °C under gentle agitation.

Techniques: Expressing, Gradient Centrifugation, Western Blot, Comparison, Immunoprecipitation, Incubation, Immuno-Electron Microscopy, Staining, Transmission Assay, Electron Microscopy

Journal: Scientific Reports

Article Title: Different populations of Wnt-containing vesicles are individually released from polarized epithelial cells

doi: 10.1038/srep35562

Figure Lengend Snippet: ( a ) Detection of Wnt11 (indicated by the closed arrow) in culture supernatant (Culture Sup.) and in P100 pellets. MDCK cells expressing Wnt11 were cultured in transwells, and soluble Wnt11 in the culture medium was concentrated by use of Blue Sepharose. Then, concentrated samples were suspended in distilled water; and P100 pellets were prepared as described for Wnt3a. Arrowheads indicate the position of Wnt11 protein. Upper bands shown in culture sup. are non-specifically cross-reactive. ( b ) Sucrose density-gradient centrifugation analysis of Wnt11. The P100 pellet prepared from the apical side of Wnt11-expressing MDCK cells was further fractionated by sucrose density-gradient as was shown in . Wnt11, Flo2, and CD63 were analyzed by Western blotting. (Full-length and smaller forms of CD63 are indicated by the closed and open arrow, respectively) Results shown are representative of 2 independent experiments. Full-length blots are presented in .

Article Snippet: The P100 pellet isolated from apical MDCK conditioned medium was incubated with 1 × 10 5 anti-CD63 or isotype control conjugated beads in 300 μL wash buffer (PBS containing 2% exosome-depleted FCS) overnight at 4 °C under gentle agitation.

Techniques: Expressing, Cell Culture, Gradient Centrifugation, Western Blot

Journal: Scientific Reports

Article Title: Different populations of Wnt-containing vesicles are individually released from polarized epithelial cells

doi: 10.1038/srep35562

Figure Lengend Snippet: ( a ) Western blot analysis of P100 pellets to detect recoveries of Wnt3a mutated at the lipidation site, Ser209, produced by MDCK cells. The amounts of Wnt3a in S10 sup and P100 pellet from conditioned medium, as well as the amount in the cell lysate, from MDCK cells expressing the lipidation site mutant Wnt3a (S209A), in which Ser209 was substituted to Ala, were analyzed by Western blotting. Comparative analysis with samples prepared from wild-type Wnt3a-expressing MDCK cells is shown in . ( b,c ) Sucrose density-gradient centrifugation analysis of the lipidation site mutant. The P100 pellet from either the apical ( b ) or basolateral ( c ) side of MDCK cells expressing the S209A mutant of Wnt3a was subjected to continuous sucrose density-gradient as in . The amounts of Wnt3a, Flotillin2 (Flo2), CD63, and Tsg101 were analyzed by Western blotting. Results shown are representative of 3 independent experiments. Full-length blots are presented in .

Article Snippet: The P100 pellet isolated from apical MDCK conditioned medium was incubated with 1 × 10 5 anti-CD63 or isotype control conjugated beads in 300 μL wash buffer (PBS containing 2% exosome-depleted FCS) overnight at 4 °C under gentle agitation.

Techniques: Western Blot, Produced, Expressing, Mutagenesis, Gradient Centrifugation

Journal: Nature Communications

Article Title: Exosomes derived from HIV-1-infected cells promote growth and progression of cancer via HIV TAR RNA

doi: 10.1038/s41467-018-07006-2

Figure Lengend Snippet: Characterization of exosomes from HIV-infected T cells. a Transmission electron microscope (TEM) images of exosomes isolated from T-cell culture supernatants. The representative J1.1 cell exosome image is shown. Scale bar, 100 nm. b Immunoblot of CD63, CD9, and CD81 on proteins extracted from T-cell line exosomes (whole scans of blots in Supplementary Figure 1). c CD63 and CD9 immunoblot (left) and AChE assays (right) of J1.1 exosomes isolated from 2, 10, and 20 ml of culture supernatants. Error bars, ± s.d. Data shown one experiment from three biological repeats. Numbers of exosomes (#Exo) were calculated by AChE activity using a standard provided by SBI

Article Snippet: Antibody against human CD63 (clone NKI/C3, cat. no. NBP2-32829, 1:200) was purchased from Novus Biologicals (Littleton, CO).

Techniques: Infection, Transmission Assay, Microscopy, Isolation, Cell Culture, Western Blot, Activity Assay