Nelson RW , Gumbiner BM .

GM17948-02 NIGMS NIH HHS , GM37432 NIGMS NIH HHS , NCI-P30-CA-08784 NCI NIH HHS , R37 GM037432 NIGMS NIH HHS , F32 GM017948 NIGMS NIH HHS , R01 GM037432 NIGMS NIH HHS

	Figure 1. A cell-free assay system for β-catenin signaling. 40 min after fertilization, cleavage blockade of Xenopus embryos is brought about by a gentle spin over a Ficoll cushion (500 g), and then are incubated until nonblocked sibling embryos reach stage 8. β-Catenin, lithium, or other components are then introduced into embryonic cytoplasm by a 16,000 g spin crush, followed by an incubation to allow for induction of target genes in underlying nuclei.
	Figure 2. Cleavage blockade reduces the normal expression of dorsal marker genes. Activation of β-catenin target genes and EF-1α (loading control) was assessed by RT-PCR in whole developing embryos (WE) and in coenocytic embryos (Coen.) at stage 7 (pre-MBT) and stage 9 (post-MBT).
	Figure 3. Target gene induction and β-catenin dose responsiveness in the cell-free assay system. A, Increasing amounts of β-catenin protein (as indicated) were added to 20 crushed embryos under standard cell-free assay conditions. RT-PCR (upper panel) analysis of Siamois expression revealed a dose-dependent induction. Western blot analysis of total lysate and ConA-Sepharose– treated (glycoprotein depleted) lysate was carried out using antibodies to the NH2-terminal region of β-catenin (lower two panels). Endogenous and free β-catenin show different electrophoretic mobility, due to the presence of a 6× His tag on the exogenous protein. Incubation of extracts with ConA-Sepharose effectively depleted C-cadherin below detectable levels (data not shown). B, In addition to Siamois, addition of β-catenin (1.5 μg/20 embryo condition) induces expression of XTwin and Xnr3 mRNA, as demonstrated by RT-PCR.
	Figure 4. β-catenin induces a subset of dorsal marker genes in a protein synthesis independent manner. The standard cell-free assay (± β-catenin protein) was carried out with and without 5 μg/ml (cytoplasmic concentration) cycloheximide, as indicated. This dose of cycloheximide effectively inhibits protein translation in crushed embryos, as is evidenced by 35S incorporation (right). RT-PCR analysis (left) revealed that β-catenin induced Siamois, XTwin, Xnr3, and Cerberus in the presence or absence of protein synthesis. Other genes that were analyzed were not induced by β-catenin in any condition. The lane marked WE St. 9 represents an RT-PCR reaction conducted on cDNA derived from normal embryos at stage 9 (at the termination of the assay).
	Figure 5. Lithium induction of Siamois mRNA expression. A, Lithium was introduced at a final cytoplasmic concentration of 16 mM in crushed embryo cytoplasm under standard assay conditions. RT-PCR analysis of gene expression reveals that this concentration of lithium induces Siamois in the presence or absence of protein synthesis. EF-1α serves as a loading control. Protein synthesis was blocked by inclusion of cycloheximide in the crushed embryo cytoplasm (final cytoplasmic concentration, 5 μg/ml), as is demonstrated by the loss of metabolically labeled protein bands in the right panel. B, RT-PCR analysis of Siamois expression reveals that addition of 2 μl of 1 mg/ml recombinant C-cadherin intracellular domain protein (CT) effectively blocks induction by exogenous β-catenin (left) or lithium (right), whereas equivalent amounts of the membrane-proximal portion (P) of the intracellular domain of C-cadherin (lacking catenin binding sites) has no effect (right and left).
	Figure 6. Lithium does not affect the levels of endogenous or exogenous β-catenin. A, An anti–β-catenin probed Western blot shows levels of endogenous total or non-ConA precipitable (post-ConA) β-catenin, with or without lithium induction. B, Addition of lithium did not affect the stability of small, nonsignaling amounts of added recombinant β-catenin. 7.5 ng of β-catenin protein was added to assay tubes in the presence or absence of added lithium. Total 1% NP-40 lysate was analyzed by Western blot using antibodies against β-catenin. Exogenous and endogenous β-catenin are distinguished by their electrophoretic mobility, as marked.
	Figure 7. Effects of proteasome inhibitors on Siamois induction. Samples treated with lithium and/or the proteasome inhibitors ALLN or MG132 were evaluated by RT-PCR using Siamois and EF1α primer sets. Proteasome inhibitors (25 μM), introduced upon crushing the embryos, did not induce Siamois and did not affect lithium induction of Siamois.

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