Copyright information:Taken from "Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/β-catenin signaling"http://www.jmolecularsignaling.com/content/2/1/12Journal of Molecular Signaling 2007;2():12-12.Published online 25 Oct 2007PMCID:PMC2211464. immunoprecipitated with Anti-Dvl2 antibody. Bound proteins were resolved by SDS-PAGE, immunoblotting, and made visible by staining with either anti-Dvl2 or anti-PP2A C-subunit antibodies. Dvl2 and PP2AC association is displayed as "fold" (time = 0, set to "1"). Representative blots are displayed. The results are shown as mean values ± S.E. from 5 independent experiments., F9 cells expressing Rfz1 were stimulated with purified Wnt3a for the indicated time. Cells were washed with PBS and lysed. Cell lysates were applied to a small Sephadex G-50 column to remove small molecular substances that interfere with the assay, and PP2A activity determined. Results were shown as the mean values ± S.E. from 8 independent experiments

Boogaard, G.J.P.M., van den

Janssen, L.H.J.

Wild, J.P.J.

Polderdijk, J.J.

Berkenbosch, A.C.

Dutch

Copyright information:Taken from "Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/β-catenin signaling"http://www.jmolecularsignaling.com/content/2/1/12Journal of Molecular Signaling 2007;2():12-12.Published online 25 Oct 2007PMCID:PMC2211464.g Rfz1 were treated with Wnt3a for 8 hr in the absence or presence of OA, added 60 min prior to stimulation with Wnt3a. Cells were lysed and the Lef/Tcf-sensitive gene transcription was assayed in samples of cell lysates using the M50 luciferase gene reporter. The results showed mean values ± S.E. that were obtained from five separate experiments. Statistical significance is noted (*, < 0.001; **, < 0.05). , OA enhanced the time-course of activation of Lef/Tcf-sensitive transcription in response to stimulation by Wnt3a. F9 cells expressing Rfz1 were treated without (open circle) or with (closed circle) OA and Wnt3a added for a 10 hr stimulation. Cells were harvested at indicated time points and disrupted. The Lef/Tcf-sensitive gene transcription was assayed. The results shown are mean values ± S.E. obtained from four separate experiments. Statistical significance is denoted (*, for < 0.005). , effects of OA on the cellular abundance of Wnt/β-catenin signaling elements in response to long-term (0–10 hr) stimulation by Wnt3a. The cellular content of β-catenin, Dvl2, GSK3β, and GAPDH (as a control) were established in F9 cells expressing Rfz1 and stimulated with Wnt3a, in the absence or presence of OA, for 0–10 hr. The results shown are mean values ± S.E. obtained from four separate experiments. Representative blots are displayed

Noriko Yokoyama (70608)

Craig C Malbon (70609)

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Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/β-catenin signaling-9

Copyright information:Taken from "Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/β-catenin signaling"http://www.jmolecularsignaling.com/content/2/1/12Journal of Molecular Signaling 2007;2():12-12.Published online 25 Oct 2007PMCID:PMC2211464.s were fractionated to the plasma membrane (PM), cytoplasm (CY) and nuclei (NU), as described in the . Subcelluar fractions obtained from control (time = 0) and Wnt3a-stimulated cells were analyzed by immunoblotting with anti-PP2A C-subunit antibody. PM (blue line), CY (pink line) and NU (green line) fractions are displayed. Stained protein bands were quantified and the values are presented as "fold of zero time point". Bottom of immunoblots are stained with antibodies against the following subcellular fraction marker proteins: Na-K-ATPase (plasma membrane), GAPDH (cytoplasm) and fibrillarin (nuclei), respectively. The results are shown as mean values ± S.E. from 5 or more independent experiments. Representative blots are displayed, as is the quantitative analysis of cellular content extracted from a compilation of data from the separate experiments (graphs)

Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/β-catenin signaling-6

Copyright information:Taken from "Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/β-catenin signaling"http://www.jmolecularsignaling.com/content/2/1/12Journal of Molecular Signaling 2007;2():12-12.Published online 25 Oct 2007PMCID:PMC2211464.t (time + 0) or with Wnt3a for the indicated time periods. At indicated time points, cell cultures were harvested, disrupted, and subjected to subcellular fractionation to plasma membrane (PM), cytoplasm (CY) and nuclei (NU) fractions, as described in the . Samples of each fraction were subjected to protein determination, SDS-PAGE, and the resolved proteins blotted and stained with antibodies specific for GSK3β and with antibodies specific for Ser9 phospho-GSK3β. shows the quantitative analysis of the blots for Wnt3a alone (blue line) or Wnt3a in the presence of OA (pink line). The data are displayed as fold of control (time zero set to 1). displays immunoblots stained with GSK3β and phospho-GSK3β (Ser 9) antibodies as well as antibodies specific for subcellular fractions, (., marker proteins). The results shown are derived from a single experiment, representative of two additional experiments

Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/β-catenin signaling-5

Copyright information:Taken from "Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/β-catenin signaling"http://www.jmolecularsignaling.com/content/2/1/12Journal of Molecular Signaling 2007;2():12-12.Published online 25 Oct 2007PMCID:PMC2211464. Wnt3a in the presence or absence of OA for the indicated time periods. Cells were harvested and fractionated to subcellular fractions highly enriched in plasma membrane (PM), cytoplasm (CY), or nuclei (NU), as described in the . Each fraction was separated by SDS-PAGE and the resolved protein transferred to blots stained with anti-Axin, anti-Dvl2 or anti-β-catenin antibodies. The enrichment of the subcellular fractions was established by staining immunoblots with antibodies to well known protein markers for PM, CY, and NU subcellular fractions. displays the quantitative analysis of the blots for Wnt3a alone (blue line) or Wnt3a in the presence of OA (pink line). The data are displayed as the mean values "% of control" (., t = 0 then set to 100%). displays immunoblots. The results shown are derived from a single experiment, representative of two additional experiments

Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/β-catenin signaling-4

Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/β-catenin signaling-8

Copyright information:Taken from "Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/β-catenin signaling"http://www.jmolecularsignaling.com/content/2/1/12Journal of Molecular Signaling 2007;2():12-12.Published online 25 Oct 2007PMCID:PMC2211464.2A C- subunit for 48 hr, or transfected to express SV40 small antigen for 48 hr. Cells were washed with PBS twice and lysed. Cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting, blots stained with anti-Axin, anti-β-catenin, anti-Dvl2, anti-GSK3β, anti-p-Ser (9)-GSK3β, anti-PP2A C or anti-GAPDH antibody. The relative amounts of the proteins in each fraction were established by densitometry, as described in . The relative abundance of each signaling molecule in the untreated cell, whole-cell extract was set to "1". The results are shown as mean values ± S.E. from 8–10 independent experiments. displays the representative immunoblots, stained for Axin, β-catenin, GSK3β and p-Ser (9)-GSK3β

Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/β-catenin signaling-2

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