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. 2017 Aug 22;27(12):1422–1440. doi: 10.1038/cr.2017.107

Figure 3.

Figure 3

Twa1 facilitates Wnt-induced β-catenin nuclear accumulation through its CRA domain. (A) GST pull-down analysis of purified His-β-catenin and wild-type or mutant GST-Twa1 in vitro. LisH, Lis1 homology domain; CTLH, C-terminal to LisH domain; CRA, CT11-RanBPM domain. (B) HEK-293 cells transfected with the indicated constructs were subjected to co-IP and subsequent western analysis. (C-F) HEK-293 cells infected with sh-Twa1- or sh-ctr-containing lentiviruses were transfected with RNAi-resistant wild-type or mutant human Twa1 constructs, treated with Wnt3a-CM or Ctr-CM, and then processed for immunoflouresence (C), western blotting (D), luciferase reporter assays (E) and Wnt target gene expression analysis (F). Scale bars, 10 μm. (G-J) HEK-293 cells transfected with wild-type or mutant Twa1 constructs containing NLS sequence were subjected to immunoflouresence (G, H), luciferase reporter assays (I) and Wnt target gene expression analysis (J). DNA was visualized with DAPI (blue). Bars, 10 μm. Quantitative data are expressed as the mean ± SEM (at least three independent experiments). ns, not significant. *P < 0.05 and **P < 0.01, Student's t-test.