Xenbase Image ID: 49520

Figure 2. Wnt Signaling Causes Sequestration of GSK3 inside Multivesicular Endosomes (A) GSK3 kinase activity was decreased by 66% 5% by Wnt3a treatment and was recovered after membrane solubilization with 0.1% Triton X-100 in Digitoninpermeabilized L cells. LiCl inhibition shows that the radioactive assay was specific for GSK3. Data are from two independent experiments using untransfected L cells. GSK3b and a-tubulin provide loading controls. (B) GSK3b becomes protected from Proteinase K after Wnt3a treatment, but only in the absence of Triton X-100 (lanes 3). Similar results were obtained in five independent experiments (untransfected L cells). All samples were permeabilized with Digitonin, which causes leakage of 37% of the endogenous GSK3 (three independent experiments). (C and D) Cryoimmunoelectron microscopy demonstrating relocation of endogenous GSK3b into MVBs (arrows) after Wnt3a treatment in untransfected 3T3 cells. (E) GSK3-GFP localized in MVBs (white arrows) in CA-LRP6 transfected HeLa cells but was cytosolic in control cells lacking CA-LRP6 transfection. (H) Rab5-QL-DsRed forms giant endosomes (arrows), whereas GSK3b-GFP remains uniformly distributed in the cytosol (n = 100). (K) In the presence of CA-LRP6, GSK3-GFP is translocated inside Rab5-QL giant multivesicular endosomes (see arrows) in 77% 9%, n = 80, of cotransfected cells. Note that GSK3 becomes depleted from cytosol. Data are represented as mean SEM. Image published in: Taelman VF et al. (2010) Copyright © 2010. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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