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Fig. 6
waif1a Interacts with LRP6 and Inhibits Wnt3a-Induced LRP6 Internalization without Affecting LRP6 Phosphorylation
(A) Waif1a-GFP and Waif1/CD44/Waif1-GFP coimmunoprecipitate with LRP6-HA, but not LDLR-HA in HEK293T cells.
(B) Waif1a-GFP does not block Wnt3a-induced phosphorylation of LRP6 at S1490 in HEK293T cells assayed at 8 hr poststimulation with Wnt3a CM, whereas Dkk1 overexpression does.
(C–E) Subcellular localization of Flag-LRP6 and S1490 phosphorylated LRP6 in HEK293T cells treated with Wnt3a CM (C) and in cells transfected with Waif1a-GFP (D) or Waif1c-GFP (E). Scale bars represent 5 µm.
(F) Quantification of Wnt3a-induced Flag-LRP6 internalization. membrane, clear localization to the cell surface, few puncta in cytosol; membrane + cytosol, localization of puncta to cell surface and puncta in cytosol; cytosol, no cell surface signal, more than 20 puncta in the cytosol. n = 110 Flag-LRP6, -Wnt3a; n = 105 Flag-LRP6, +Wnt3a; n = 107 Flag-LRP6+Waif1a-GFP, +Wnt3a; n = 114 Flag-LRP6+Waif1c-GFP, +Wnt3a.
(G) Cell surface LRP6 detected by biotinylation and total LRP6 in control or Waif1a-Flag transfected cells treated with Wnt3a. Lower panel shows ratio of cell surface LRP6 (top) to total LRP6 (middle) with values at zero Wnt3a set to 100%. Means ± SEM from three independent experiments.
See also Figure S6.
Reprinted from Developmental Cell, 21(6), Kagermeier-Schenk, B., Wehner, D., Ozhan-Kizil, G., Yamamoto, H., Li, J., Kirchner, K., Hoffmann, C., Stern, P., Kikuchi, A., Schambony, A., and Weidinger, G., Waif1/5T4 Inhibits Wnt/β-Catenin Signaling and Activates Noncanonical Wnt Pathways by Modifying LRP6 Subcellular Localization, 1129-43, Copyright (2011) with permission from Elsevier. Full text @ Dev. Cell