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. 2009 Jul 10;284(36):24192–24203. doi: 10.1074/jbc.M109.023044

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© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 3. — Mal colocalization at the plasma membrane with TRAF6 requires an intact TRAF6 binding motif. HEK293 cells were cotransfected with DsRed-tagged Mal (A), MalE190A (B), or MalΔx3 (C) with TRAF6-FLAG for 48 h at 32 °C. Immunofluorescence microscopy of Z-stack (29–45) images were taken of individual cells, and three-dimensional image reconstruction was performed using Imaris software (BitPlan). Colocalization illustrates TRAF6-FLAG colocalizing with Mal-DsRed in plasma membrane ruffles (arrows, right panel; bar, 8 μm). MalE190A-DsRed and MalΔx3-DsRed mutant demonstrate plasma localization, whereas TRAF6 is diffusely distributed within the cytosol (right panel; bar, 8 μm), and minimal colocalization is observed (right panel; bar, 10 μm). All of the results are representative of three independent experiments.