J. Biomol. Scr.
A biochemical screen for identification of small-molecule regulators of the wnt pathway using Xenopus egg extracts.
Thorne, C.A., Lafleur, B., Lewis, M., Hanson, A.J., Jernigan, K.K., Weaver, D.C., Huppert, K.A., Chen, T.W., Wichaidit, C., Cselenyi, C.S., Tahinci, E., Meyers, K.C., Waskow, E., Orton, D., Salic, A., Lee, L.A., Robbins, D.J., Huppert, S.S. and Lee, E.
Notes: The authors used a biochemical assay using Xenopus egg extracts to monitor degradation levels of two Wnt pathway components, Axin and ß-catenin, and identify modulators of the Wnt pathway. ß-catenin and Axin were expressed in vitro as firefly and Renilla luciferase fusion proteins, respectively, using the TNT® SP6 High-Yield Protein Expression System and shown to behave in Xenopus extracts in a similar way to wildtype proteins, which were expressed as radiolabled proteins in rabbit reticulocyte lysate. Degradation of labeled proteins was monitored by SDS polyacrylamide gel electrophoresis and autoradiography, and degradation of luciferase fusion proteins was examined using the Dual-Glo® Luciferase Assay. Using the Xenopus extract-based assay, the authors screened chemical libraries to identify two modulators of the Wnt pathway, then confirmed this inhibition in HEK 293 cells by demonstrating a decrease in ß-catenin expression with increasing concentrations of the inhibitors. This decrease in ß-catenin-Fluc levels was measured using the Steady-Glo™ Luciferase Assay, and luciferase activity was normalized to cell viability, as determined using the CellTiter-Glo® Assay. (4181)
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