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. 2013 Dec 1;24(23):3764–3774. doi: 10.1091/mbc.E13-07-0357

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© 2013 Gon 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 6: — Complex formation between FAK, Dvl, Tiam1, and p190RhoGAP-A. (A) Lysates of Wnt5a-expressing or Wnt5a-depleted IEC6 cells cultured on Matrigel were immunoprecipitated with anti-Dvl (DIX) antibody. The immunoprecipitates were probed with anti-FAK and anti-Dvl2 antibodies. (B) The amounts of precipitated FAK and Dvl 2 shown in A were quantified by using Image J, and the ratio of the former to the latter was calculated. The ratios of the interaction of FAK and Dvl2 in Wnt5a-overexpressing or Wnt5a-depleted cells were expressed as fold changes compared with controls. (C) Flag-Dvl2 was expressed in HEK293T cells stably expressing GFP-p190RhoGAP-A, and the lysates were immunoprecipitated with anti-GFP or anti-Flag antibody. The immunoprecipitates were probed with anti-GFP and anti-Flag antibodies. (D) Myc-Tiam1 and/or HA-Dvl1 (DEP+) were expressed in HEK293T cells stably expressing GFP-p190RhoGAP-A, and the lysates were immunoprecipitated with anti-GFP or anti-Myc antibody. The immunoprecipitates were probed with anti-Myc, anti-hemagglutinin (HA), and anti-GFP antibodies. (E) Myc-Tiam1 and HA-Dvl2 were expressed in HEK293T cells stably expressing GFP-p190RhoGAP-A, and the lysates were immunoprecipitated with anti-Dvl (DIX) antibody. The immunoprecipitates were probed with anti-Myc, anti-GFP, anti-FAK, and anti-HA antibodies. (F) GFP or GFP-Dvl2 deletion mutant (1–509)–expressing IEC6 cells transfected with siDvls-2 siRNA were plated on Matrigel. Polarized cells among total cells were counted, and the percentage of polarized cells was calculated. The ratios of the percentages of polarized cells were compared with controls. Results are shown as means ± SD from three independent experiments. *p < 0.01.