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. 2013 Jun 1;24(11):1688–1699. doi: 10.1091/mbc.E12-05-0386

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© 2013 Ponik 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: — p120-catenin contributes to localizing p190RhoGAP-B to cell–cell contacts and to regulating activity of RhoA. (A) T47D cells expressing p120-catenin shRNA or the appropriate vector control plasmid were cultured in attached or floating 1.3 mg/ml collagen gels for 10 d. Phase contrast images show that shRNA against p120-catenin disrupts tubulogenesis in floating gels, as well as normal cell growth in attached collagen gels (scale bar, 100 μm). (B) In these cell lines, RhoA activity was down-regulated in T47D control cells and in p120-catenin shRNA cells cultured in compliant vs. rigid collagen gels (*compliant vs. rigid: untransfected, p = 0.0001; pRS, p = 0.0011; p120shRNA, p = 0.0464; n = 5). Of interest, p120-catenin is necessary for the proper level of RhoA activity in both compliant and rigid collagen gels, as RhoA activity is significantly elevated in p120-catenin shRNA–expressing cells compared with untransfected and vector control cells (*p120shRNA vs. untransfected, p = 0.0214; p120shRNA vs. pRS, p = 0.0141; n = 5). (C) Top, Immunofluorescence analysis of p120-catenin localization in control and p190B shRNA cells. Knockdown of p190B did not alter the localization of p120-catenin in compliant or rigid collagen gels. Bottom, analysis of p190B localization in control vs. p120-catenin shRNA cells completed after culture in compliant and rigid collagen gels. In contrast to p190B shRNA cells, knockdown of p120-catenin results in the visible loss of p190B at cell–cell contacts. (D) Western blot analysis confirmed that the total level of p190B was not altered in control, human-specific p120-catenin-shRNA or mouse p120-catenin-3A rescue cell lines. z was used as a loading control. Quantification of p190B immunofluorescence in regions of interest demonstrate a significant decrease in p190B intensity at sites of cell–cell contact in both rigid and compliant collagen gels when p120-catenin is knocked down (scale bar, 100 μm; *rigid and compliant pRS vs. p120-catenin shRNA, p = 0.004; n = 9 fields of cells in three experiments). The localization of p190B to cell–cell contacts was restored to 75% of control levels and significantly increased above knockdown levels upon rescue of p120-catenin with mouse p120CTN-3A (*rigid and compliant: p120-catenin shRNA vs. mouse-p120CTN-3A rescue, p = 0.042; n = 9 fields of cells in three experiments).