Figure 3. Comparison of Wnt ligand mediated internalization of LRP6 WT and a representative LDLRR mutant.

(A). Cell surface LRP6 expression in Wnt autocrine sarcoma cell line, U-2OS following Nystatin treatment. Low and High indicate cell confluency. For Pair Control and Nystatin, cell numbers were similar at the start of treatment. FACS analysis was performed as in Figure 2A, except that monoclonal LRP6 antibody [57] was used. The values represent the geometric mean obtained from 10,000 events counted. (B). Effect of Nystatin on cell surface LRP6 expression (i) and uncomplexed β-catenin level (ii) in Wnt autocrine A204 and A3243 human sarcoma cells [30]. Cells were pre-treated with Nystatin for 5 hours and then labelled with cell impermeable biotin. After incubation, cells were rinsed and lysed in NP-40 buffer and processed for immunoprecipitation with either Avidin conjugated beads or with GST-E-cadherin/glutathione conjugated beads. (C). 293T cells transfected with either WT or LDLRR mutant LRP6 were treated with Wnt3a for different times as indicated and then fixed in 4% paraformaldehyde and processed for FACS analyses as in Figure 2A. The mean fluorescence values obtained were normalized to the untreated sample (shown as 1). Each data point represents the mean value obtained from at least 3 independent experiments performed under similar conditions. Error bars indicate SEM. The graph is representative of three independent experiments. The p values were calculated using unpaired two-tailed t-test comparing values at each time point between the two groups. (D). Cell surface clearance of LRP6 in Wnt3a treated cells. 293T cells transfected with either WT or LDLRR mutant LRP6 were treated with Wnt3a for the times indicated. After rinsing, cells were labelled with cell impermeable biotin. Biotin was then removed, and cells were rinsed, lysed in NP-40 buffer and processed for IP with avidin-conjugated beads. Protein eluted was used for immunoblotting. The pixel values obtained for LRP6 from IP were normalized to those obtained for LRP6 in WCL and represented as relative ratios. Each data point is mean value obtained from at least three independent experiments run at similar conditions. Error bars indicate SEM. The graph is a representative of three independent experiments. The p values were calculated using unpaired two-tailed t-test comparing values at each time point between the two groups. (E). LRP6 internalized after Wnt3a treatment of 293T cells. This experiment was performed as in Fig.3D, except that cells were biotin labelled prior to Wnt3a treatment. Following Wnt3a treatment for the time intervals indicated, cells were rinsed and incubated with glutathione on ice to remove any remaining biotinylated cell surface proteins prior to lysis and IP. Each data point in the graph is mean value obtained from at least three independent experiments performed under similar conditions. Error bars indicate SEM. The graph is a representative of three independent experimental repeats. The p values were calculated using unpaired two-tailed t-test comparing values at each time point between the two groups. (F). Comparison of LRP6 internalized after 1 hr of Wnt3a treatment of 293T cells expressing WT or various LDLRR mutants. This experiment was performed as in Figure 3E. Western blots were processed using Photoshop to adjust brightness/contrast and cropped to show all important bands.