Ates were analysed by Western Blot for the levels of β-catenin. Pathway activation is indicated by the increase in cytosolic β-catenin upon Wnt-3a treatment seen only in the parental HEK293 cells whereas in the SFPR-4-expressing cells this effect was abolished. conditioned medium supernatant (CM) prepared from the HEK 293 cells expressing SFRP-4(HA tagged) after treatment with 5 μl Wnt-3a for 4 hours in the above experiment, was immunoprecipitated with an anti-HA-tag antibody, electrophoresed, Western blotted and, firstly, detected with an anti-HA-tag antibody. The first lane shows the precipitated SFRP-4 containing the HA tag from the CM. The middle lane shows the presence of the same protein in the total cell lysate (TCL) of the same cells as a control. The membrane was then re-probed with affinity-purified anti-Wnt-3a antibody, showing binding of Wnt-3a to the immunoprecipitated SFRP-4(HA tagged) from the conditioned medium supernatant (first lane), but not in the total cell lysate (middle lane). In addition, a sample of TCL from L cells expressing Wnt-3a (Fig. 8b, last lane), which had not been subjected to immunoprecipitation, served as a control demonstrating the detection of Wnt-3a protein.<p><b>Copyright information:</b></p><p>Taken from "SFRP-4 abrogates Wnt-3a-induced β-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of mammary differentiation"</p><p>http://www.jmolecularsignaling.com/content/3/1/10</p><p>Journal of Molecular Signaling 2008;3():10-10.</p><p>Published online 2 May 2008</p><p>PMCID:PMC2412851.</p><p></p
