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. 2017 Jan 1;28(1):41–53. doi: 10.1091/mbc.E16-07-0557

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© 2017 Pronobis et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 4: — The APC:Axin Chimera reduces βcat as efficiently as wild-type APC plus Axin. (A) Schematic, APC and Axin constructs plus Chimera. Top, wild-type APC and Axin with essential regions indicated. Middle, mutants incorporating essential regions alone. Bottom, essential regions were combined into a single polypeptide to create the Chimera. (B–H) Immunofluorescence, GFP-tagged or RFP-tagged wild-type or mutant constructs misexpressed in SW480 cells and stained for βcat via antibody. Arrows point to transfected cells. Close-ups of a transfected cell are shown to the right to reveal which constructs retain βcat in puncta. (B) Wild-type GFP-APC2 reduces βcat levels. (C) Wild-type GFP-Axin also reduces βcat levels, but some βcat remains in puncta (arrowheads, close-up). (D) Coexpressing GFP-APC and Axin-RFP effectively reduces βcat levels. (E) GFP-APC2ARB, which consists of APC’s three essential regions, can moderately reduce βcat levels, but βcat remains higher than is seen after wild-type APC2 transfection. (F) Axinβcat-DIX-RFP also moderately reduces βcat levels, and detectable βcat remains in the puncta (arrowhead, close-up). (G) Coexpressing APC2ARB and Axinβcat-DIX does not further decrease βcat levels, and βcat remains in puncta (arrowhead, close-up). (H) Expressing the GFP-Chimera leads to strong reduction of βcat, and no βcat is seen in puncta (arrowhead, close-up). (I, K, M) Quantification, βcat fluorescence intensity in SW480 cells transfected with indicated constructs, normalized to untransfected cells. Ten cells each in three independent experiments were measured. (J, L, N) Quantification of Wnt-regulated transcription in SW480 cells (TOPflash activity, normalized to untransfected cells). Three triplicates were measured in three independent experiments. (I) Axin cannot reduce βcat levels as effectively as APC2 or APC2 + Axin. (J) Axin cannot inhibit Wnt-regulated transcription as effectively as APC2 or APC2 + Axin. (K) Mutants carrying only the essential regions of APC2 or Axin only moderately reduce βcat levels, whereas covalently linking the essential regions of APC and Axin into the Chimera increases βcat reduction. (L) The Chimera strongly inhibits Wnt-regulated transcription. (M) The Chimera reduces βcat levels better than wild-type Axin. (N) Wnt-regulated transcription is as effectively inhibited by the Chimera as it is by wild-type APC2 + Axin or APC2. Student’s t test was used. (O) APC2, Axin, and the Chimera are not dosage dependent in βcat degradation. GFP levels (reflecting expression level of APC or Axin construct) vs. βcat signal in individual cells expressing each construct. βcat signal is normalized to nearby untransfected cells. A set of individual values of untransfected cells shows the degree of variability among cells in the same population. Thirty cells total for each condition. Both the Chimera and APC2 are more effective at reducing βcat than is Axin over a wide range of expression levels. (P–R) Immunoblot, expression levels of indicated constructs. aPKCγ is a loading control. Relative expression levels vary somewhat from experiment to due to transfection efficiency. (S) α-Catenin coimmunoprecipitates with the Chimera. Left, cell lysates from cells expressing the indicated constructs. γ-Tubulin serves as a loading control. Right, anti-GFP immunoprecipitates from cells expressing the indicated constructs. Bottom, effectiveness of antibody pull down.