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Fig. 4. β-catenin-uncoupled E-cadherin is targeted to lysosomes. (A) Intracellular accumulation of EC81 does not depend on clathrin-mediated endocytosis. MDCK cells stably expressing EC81 were transfected with (upper panel) wild-type or (lower panel) dominant-negative dynamin and analyzed for their transient expression. Asterisks indicate cells expressing GFP-tagged dynamin. (B) Newly synthesized Ecad is expressed at the cell surface but EC81GFP is not. MDCK cells stably expressing Ecad or EC81GFP were incubated with 10 µM cycloheximide for 12 hours to deplete Ecad or EC81GFP from the protein synthesis pathway and the cell surface. Cycloheximide was washed out, and cells were returned to normal medium for the indicated times before fixation. Cell-surface localization of Ecad is already detectable after 4 hours, increasing over time. EC81GFP was never detected at the surface. (C) ESA and EC81 decreased upon treatment with cycloheximide. Cells were incubated for 2 hours in the presence or absence of 10 µM cycloheximide (CHX) and then subjected to immunoblot analysis using anti-HA antibody. (D) Effect of lysosome and proteasome inhibitors on ESA and EC81 stability. Cells were incubated for 3 hours with the lysosome inhibitor chloroquine (CQ, 200 µM) or the proteasome inhibitor MG132 (MG, 10 µM). Cell lysates were prepared and analyzed by western blotting using anti-HA antibodies to detect ESA and EC81. (E) Quantitative analysis of C and D, indicating that ESA and EC81 levels are increased in lysosomes in cells treated with chloroquine. (F) Accumulation of EC81 in lysosomes after chloroquine treatment. Cells were incubated for 3 hours in the presence (+CQ) or absence (–CQ) of chloroquine and processed for immunofluorescence staining with anti-HA. Images show a marked accumulation of EC81 after treatment.
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