Figure 5. Role of LRP6 ubiquitination on Wnt3a mediated internalization.

(A&B). Endogenous LRP6 protein and Axin2 mRNA expression in ubiquitin knockdown or ubiquitin rescued Wnt autocrine U-2OS sarcoma cells. U-2OS cells engineered to be devoid of endogenous ubiquitin and expressing exogenous WT ubiquitin under Dox inducible promoter were used [35]. 72hrs post induction with Dox, cells were processed either for immunoblotting (A) or for real-time PCR (B). Axin2 mRNA expression was normalized to TATA Box Binding Protein (TBP). Each bar represents the mean of triplicates from one experiment, and error bars are SEM. The entire experiment was repeated twice with similar results. The p values were calculated using unpaired two-tailed t-test comparing values of “induced” in the two groups. (C). Wnt signaling activity of a cytoplasmic lysine mutant LRP6. All lysines present in the cytoplasmic tail of LRP6-WT were mutated to arginine, and the resulting mutant, designated KR10, or WT LRP6 was transfected in 293T cells stably expressing TCF and Renilla luciferase. 24 hrs later, the cells were treated with different concentrations of Wnt3a conditioned medium as indicated and processed for dual luciferase assay (top) or immunoblotting (bottom). Treated samples are represented relative to the control untreated sample in both WT and KR10 groups. Each bar represents the mean of triplicates from one experiment and error bars are SEM. The entire experiment was repeated twice. The p values were calculated using unpaired two-tailed t-test comparing values of treated samples with the control untreated in each group. (D). Cell surface clearance of cytoplasmic lysine mutant LRP6. 293T cells transfected with wild type or lysine mutant LRP6 were labeled with biotin, then treated with Wnt3a for 1 hr and processed as in Figure 3D. c-MET was used as a cell surface control protein. The graph on the right represents quantification of the pixels from the immunoblots as described in Figure 3D. Bars represent mean values of three independent experiments conducted under similar experimental conditions and error bars are SEM. The p values were calculated using unpaired two-tailed t-test comparing values of treated and untreated samples in each group. (E). Internalization of LRP6 KR10 mutant. 293T cells transfected with WT or KR10 were labeled with biotin, then treated with Wnt3a 1hr and processed as in Figure 3E. The graph was generated as in Figures 3E & 5D. (F). Interaction of the LRP6 KR10 mutant with Itch. 293T cells transfected with wild type Itch were replated and 24hrs later, transfected with LRP6 WT or KR mutant. 36hrs later the cells were treated with Wnt3a for 1hr and processed for IP and immunoblotting as in Figure 4A. Last lane:IP with IgG Control; All other lanes: IP with Flag The experiment was repeated twice with similar results. Western blots were processed using Photoshop to adjust brightness/contrast and cropped to show all important bands.