Figure 3

i27-Exosomes express membrane-bound p28, Ebi3 and CD81. (A) B-1a or CD19⁺ B-2 cells from mouse peritoneal cavity or spleen were stimulated with LPS or anti-IgM/anti-CD40 in exosomes-depleted medium for 72hrs (n=3-4/group). Exosomes in supernatant were purified and protein content in 5x10⁶ exosomes was quantified by BCA protein assay (top panel) and amount of IL-27 secreted (pg/50µg exosome protein) was determined by ELISA (bottom panel) (n=3-4/group). (B-D) Exosomes isolated from supernatant of the BCR-activated B-1a cells were subjected to qPCR analysis (B) (n=6/group), Western blot analysis (C) (n=1/group) or reciprocal immunoprecipitation analysis (D). Controls in these analyses are exosome-depleted supernatant of B1a cell cultures. (E) Cell lysates and exosomes from activated B1a or B-2 cells were analyzed for IL-35 or IL-27 by ELISA(n=3/group). (F) For localization of IL-27 (p28 and Ebi3) and CD81 expression on the i27-Exosome membrane, the exosomes were captured with Tetraspanin Exo-Flow Capture Kit consisting of 9.1 µm diameter magnetic beads, stained with antibodies without fixation or permeabilization and then analyzed by confocal microscopy. The data are presented as the mean ± SD of at least three determinations, *p<0.05; **P < 0.01; ***P < 0.001; ****p<0.0001.