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. 2014 Oct 1;25(19):2894–2904. doi: 10.1091/mbc.E13-09-0512

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© 2014 Nava, Kamekura, Quirós, 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 2: — IFNγ promotes the association of β-catenin with 14.3.3ζ. (A) Association of β-catenin with 14.3.3ζ was analyzed by coimmunoprecipitation assays. 14.3.3ζ and control immunoglobulin G (IgG) were immunoprecipitated from fresh lysates obtained from SW480 cells, control or treated with IFNγ for 1 h. 14.3.3ζ was immunoprecipitated from IECs isolated from murine intestinal mucosa exposed for 2 h to vehicle (MSA), IFNγ, and TNFα. Immunoprecipitates were blotted for β-catenin, pβ-cat552, and 14.3.3ζ. Densitometric analysis of β-catenin, pβ-cat552, and 14.3.3ζ. (B) The effect of 14.3.3ζ on β-catenin stabilization was analyzed in CHO cells. Confluent monolayers of CHO cells were transfected with 0.1–0.2 μg/ml β-catenin–expressing vector in presence of increasing concentrations of a 14.3.3ζ-expressing vector (arrow). Cell lysates were collected in RIPA lysis buffer and equal amounts of proteins loaded and analyzed by Western blotting. Actin was used as a loading control. (C) The effect of IFNγ and 14.3.3ζ (arrow) overexpression on endogenous β-catenin stability was determined by Western blot in CHO cells. Relative densitometric values were normalized with respect to the controls. p120 catenin was used as a loading control. (D) The effect of 14.3.3ζ expression on β-catenin transactivation was analyzed by TOPflash assays. SW480 cells were transfected with a vector expressing 14.3.3ζ or siRNA targeting 14.3.3ζ and luciferase expression determined. The cellular distribution of β-catenin (E) and 14.3.3ζ (F) was analyzed by immunofluorescence in SW480 cells that were exposed to vehicle (Ctl) or IFNγ for 12 h. Nuclei are blue. Bar, 10 μm.