SFRP-4 abrogates Wnt-3a-induced β-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of mammary differentiation-8

of Dvl-3. Following treatment with Wnt-3a for the indicated times, cells were lysed with Triton X-100 Lysis Buffer and cell extracts were subjected to Western blot analysis. Hyperphosphorylation of Dvl-3 is evident by a consistent relative increase in the intensity of the upper band of the doublet. Alkaline phosphatase treatment caused the loss of the upper band (data not shown).<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

SFRP-4 abrogates Wnt-3a-induced β-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of mammary differentiation-4

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

SFRP-4 abrogates Wnt-3a-induced β-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of mammary differentiation-2

Urified Wnt-3a-treated for 2 and 4 hours. At both time points cells transfected with the Dkk-1 plasmid fail to induce accumulation of cytoplasmic β-catenin upon Wnt-3a treatment, as detected by Western Blot. In contrast, Mock transfected cells show an elevation in the level of β-catenin upon Wnt-3a treatment. The detection of β-actin served as a loading control. Parallel plates were treated as above and lysates were collected for extraction of total RNA. mRNA is shown by Northern Blot in the cells transfected with Dkk-1. 18S and 28S rRNA bands are loading controls obtained by acridine orange staining of the gel used for the Northern Blot prior to blotting.<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

SFRP-4 abrogates Wnt-3a-induced β-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of mammary differentiation-3

-3a. Treatment with Wnt-3a induced a hyperphosphorylation of Dvl-3, an increase in Akt phosphorylation (Ser473), and raised the levels of "active" β-catenin in the absence (pBabeV, vector), but not in the presence of the SFRP-4 cells.<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

SFRP-4 abrogates Wnt-3a-induced β-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of mammary differentiation-7

Ombinations of growth factors and lactogenic hormones. Lines 31E +30F co-cultures containing either pBabeVector or pBabeSFRP-4-infected Line 31E cells were compared. Most notable is that Wnt-3a is capable of completely suppressing the differentiation to β-casein expression stimulated by dexamethazone-insulin-prolactin (DIP). However, co-cultures containing Line 31E cells infected with SFRP-4 show definite β-casein induction in spite of Wnt-3a treatment. Ribosomal RNA was stained with acridine orange as a loading control.<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

SFRP-4 abrogates Wnt-3a-induced β-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of mammary differentiation-0

of Dvl-3. Following treatment with Wnt-3a for the indicated times, cells were lysed with Triton X-100 Lysis Buffer and cell extracts were subjected to Western blot analysis. Hyperphosphorylation of Dvl-3 is evident by a consistent relative increase in the intensity of the upper band of the doublet. Alkaline phosphatase treatment caused the loss of the upper band (data not shown).<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

SFRP-4 abrogates Wnt-3a-induced β-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of mammary differentiation-6

Ulin + epidermal growth factor (EGF). Co-cultures, made with either pBabeVector or pBabeSFRP-4-infected Line 31E cells together with Line 30F cells, were maintained in 3% serum-containing medium and compared in their responses to Wnt-3a. With Line 31E-Vector cells (31E-V) in co-culture, either Wnt-3a alone or insulin + EGF (Ins+EGF) treatments elicit stimulation of (CD1) mRNA expression, whereas (Cx43) expression responds only to Wnt-3a, and is strongly inhibited by EGF+insulin. A response to Wnt-3a was not seen; however, when co-cultures were made using Line 31E cells expressing pBabeSFRP-4 (31E-SFRP4), up-regulation of but not was prevented. However, mRNA up-regulation is reduced compared to the co-cultures containing Line 31E-Vector cells. Residual ribosomal RNA (loading control) was stained with acridine orange.<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