Wnt3a associates with the liposomal surface.

<p>(A) The rate of Wnt activity partitioning into the liposomal pellet is shown; initially, all Wnt activity (blue lines) is found in the supernatant but within 30 min, the majority of Wnt activity is associated with the liposomal pellet and by 6 h, 90% of Wnt activity is found in the liposomal pellet. (B) Immunoblot analysis using Wnt3a antibody shows that similar to the Wnt activity, initially the majority of the Wnt is found in the supernatant (supnt); within 30 min majority of the majority of Wnt3a segregates into the liposomal pellet and by 6 h 90% of the protein is associated with the liposomal pellet. (C) Following ultra-centrifugation, the majority of the protein is found in the liposomal pellet. (D) Wnt activity (blue bar) is also found in the pellet. Although some Wnt3a is found in the aqueous supernatant, it is inactive (figure C). (E) Sucrose density gradient centrifugation and phospholipid quantification assay (orange line), demonstrate that PBS liposomes migrate to higher density fractions. (F) Sucrose density gradient centrifugation, phosphatidyl choline lipid quantification and Wnt reporter assay, demonstrate that Wnt activity (blue line), the lipids (orange line), co-fractionate on a sucrose density gradient. (G) Immunoblotting analyses using Wnt3a antibody show that Wnt co-migrates with fractions showing maximum Wnt activity and lipid concentration. (H) The stability of L-Wnt3a is measured. At 4°C, L-Wnt3a retains >80% of its activity after extended storage. (I) An anti-Wnt3a immunoblot reveals no evidence of degradation products of L-Wnt3a after extended storage. Data are mean ±SEM from, or are representative of, at least three independent replicates.