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. 2013 Feb 9;288(12):8289–8298. doi: 10.1074/jbc.M112.433185

FIGURE 4.

FIGURE 4.

NEDD4L attenuates Wnt/β-catenin signaling. A, wild-type NEDD4L, but not CA or WW3 NEDD4L mutants, attenuates Wnt3a-induced Topflash reporter expression. After transfection with the Topflash luciferase reporter with or without FLAG-NEDD4L variants for 36 h, HEK293T cells were treated with control medium or Wnt3a conditioned medium (CM) overnight and then luciferase activity was measured. The pRL-TK Renilla reporter was co-transfected to normalize transfection efficiency. The experiment was performed in triplicate, and data were represented as the mean ± S.D. after normalized to Renilla activity (**, p < 0.01). B, NEDD4L decreases wild-type Dvl2, but not AY mutant Dvl2, induced Topflash reporter activity. The experiment was performed similarly as in A except that Dvl2 was transfected to activate the reporter activity. C, NEDD4L has no effect on LiCl-induced Topflash reporter activity. The experiment was performed similarly as in A, except that 20 mm LiCl was used to activate the reporter activity. D, NEDD4L has no effect on β-catenin (S37A)-induced Topflash reporter activity. The experiment was performed similarly as in A, except that β-catenin (S37A) was transfected to activate reporter activity. E, NEDD4L knockdown increases Topflash reporter activity. The experiment was performed as in A, except that various plasmids and nonspecific (NS) or NEDD4L shRNAs were transfected for 60 h. FLAG-NEDD4L R1 and R2 were resistant to NEDD4L shRNA1 and shRNA2, respectively. F, NEDD4L shRNAs have no effect on expression of shRNA-resistant NEDD4L. HEK293T cells transfected in E were harvested for immunoblotting (IB) with anti-FLAG antibody. Tubulin served as a loading control. The asterisk indicates a nonspecific band. G–I, NEDD4L knockdown increases the expression of AXIN2, DKK1, and C-MYC. The experiments were performed similarly as in Fig. 3D. J, NEDD4L overexpression decreases the β-catenin level. After transfection with wild-type or CA FLAG-NEDD4L for 40 h, HEK293T cells were treated with control medium or Wnt3a condition medium (CM) for 6 h and then harvested for immunoblotting. Tubulin served as a loading control. K, NEDD4L knockdown increases the β-catenin level. The experiment was performed as in J, except that nonspecific or NEDD4L shRNAs were transfected for 60 h.