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. 2009 Jul 30;284(39):26716–26724. doi: 10.1074/jbc.M109.039313

FIGURE 3.

FIGURE 3.

CVAK104-induced Fzd5 accumulation occurs via the clathrin-dependent pathway. A, 293 cells were transfected with Fzd5-GFP and FLAG-CVAK104. At 48 h after transfection, cells were subjected to an internalization assay and visualized by direct observation of GFP fluorescence and staining with anti-EEA1 antibody. A merged image of the signals for GFP (green) and EEA1 (red) is also indicated. Scale bar, 5 μm. B, 293 cells expressing Fzd5-GFP and FLAG-CVAK104 were pretreated with DMSO, MDC, or nystatin for 1 h and were then subjected to an internalization assay in the presence of inhibitors. Scale bar, 5 μm. C, quantification of cells with internalized Fzd5-GFP in B. All data are mean ± S.E. (n = 3). *, p < 0.01. D, 293 cells expressing Fzd5-GFP and FLAG-CVAK104 with or without Myc-Rab5 S34N were subjected to an internalization assay and then visualized by direct observation of GFP fluorescence and immunostained with anti-FLAG antibody. Scale bar, 5 μm.