Fig. 3: Effect of membrane proteins on sFRP2-induced re-secretion of GFP-Wnt3a in exosomes.

a Effect of heparinase in sFRP2-induced exosome-mediated secretion of GFP-Wnt3a. Western blot analysis of GFP-Wnt3a recovered in exosome-enriched P100 (2nd) fraction, as well as S100 (2nd) collected 24 h after treatment of exosome-depleted CS from co-culture of GFP-Wnt3a-expressing cells and sFRP2-expressing or control cells in parental or Heparinase III (HepIII) expressing HEK293 cells. b-d. Examination of involvement of receptors of Frizzled family proteins (b) and co-receptors of LRP5 and LRP6 (c, d) in the increase of exosome-mediated GFP-Wnt3a re-secretion by sFRP2. Fzd 1–10 KO and control HEK293 cells (b) or Lrp5/6 double KO and parental MDCK cells (c) were treated with exosome-depleted CS (input S100) from co-culture of GFP-Wnt3a/L cells with or without sFRP2-expressing cells. P100 (2nd) fractions from these cells were subjected to Western blotting. The amount of GFP-Wnt3a recovered in P100 pellets collected from CS of Lrp5/6 double KO or parental MDCK cells was quantified with Image J and standardized against that from parental cells treated with exosome-depleted CS from co-culture of GFP-Wnt3a/L and control HEK293 cells (d). Data are shown as mean ± s.e. p < 0.05 is considered statistically significant. p value were calculated by two-way ANOVA followed by Turkey HSD test, n = 3 independent experiments.