Skip to main content
. Author manuscript; available in PMC: 2010 Mar 2.
Published in final edited form as: Nat Cell Biol. 2008 Sep 21;10(10):1208–1216. doi: 10.1038/ncb1781

Figure 5.

Figure 5

pVHL regulates endogenous β-catenin through Jade-1. (a) β-catenin protein levels depend on VHL status in renal cancer cell lines. Cytosolic and nuclear fractions of renal-cell carcinoma cell lines were probed for endogenous β-catenin protein. One of 2 similar experiments is shown. (b) pVHL down-regulates endogenous β-catenin. Extracts from 786-O cells stably expressing empty vector (Vec) or pVHL were probed for endogenous β-catenin protein. Jade-1 and pVHL expression was detected using polyclonal Jade-1 and monoclonal pVHL antibodies. Representative immunoblot of 3 experiments. (c) pVHL knock-down increases endogenous β-catenin abundance. To confirm pVHL knock-down, pVHL was immunoprecipitated using polyclonal pVHL or control (C) antibody from untransfected (Un) 293T cells or 293T cells transfected with VHL (VHLsi) or control siRNA (Csi) oligonucleotides because endogenous pVHL abundance is low17. Cell fractions were probed for endogenous Jade-1 and endogenous β-catenin protein. Representative immunoblot of 3 experiments. (d) Differential regulation of endogenous β-catenin by wild-type pVHL and pVHL truncations that do not stabilize Jade-1. Extracts of 293T cells transfected with wild-type pVHL, pVHL del96-122 (Δ96-122), pVHL 1-143 and pVHL 1-175 were probed for pVHL, endogenous β-catenin and endogenous Jade-1 by immunoblot. One of 2 similar experiments is shown. (e) pVHL suppresses endogenous Wnt targets. WCLs of empty vector (Vec) or pVHL-expressing 786-O cell lines were probed for pVHL and endogenous LEF1 and cyclin D1. Representative immunoblot of 3 experiments. (f) Wild-type pVHL, but not pVHL del96-122, suppresses β-catenin/Tcf target genes. In vitro transcribed, capped LacZ (0.288 ng), wild-type VHL (WT) (0.194 ng) or VHL del96-122 (0.293 ng) mRNA was injected into dorsal blastomeres of Xenopus laevis embryos. Injected embryos were harvested at stage 10 for RT-PCR. Semiquantitative RT-PCR analysis of the Wnt targets Xsiamois and Xnr3 was performed in whole embryos. ODC served as control. Unin.= uninjected.