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. 2011 Mar 1;22(5):634–650. doi: 10.1091/mbc.E10-06-0514

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© 2011 Barfod et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 7: — Src inhibition leads to the accumulation of internalized membrane marker within MLCK rings. MLCK rings may represent MLCK-coated vesicular intermediates. (A) Cells transfected with GFP-MLCK (green) were treated with exogenous FM4-64X (red) and fluorescently labeled CTXB (blue) during hypotonic exposure in the presence of PP2 (10 μM) for 1 min before fixation. Arrows mark distinct MLCK and FM4-64X membrane dye rings. (B) Stills from live imaging of cells transfected with GFP-MLCK (green) taken at the base and treated with PP2 (10 μM) during hypotonic treatment in the presence of exogenous FM4-64X. An arrow marks an MLCK ring that appears to encircle internalized membrane marker. (C) Cells transfected with GFP-MLCK (green) were treated with exogenous FM4-64X (red) and fluorescently labeled CTXB (blue) during hypotonic exposure in the presence of PP2 (10 μM) for 5 min before fixation. An arrow marks an MLCK ring that appears to encircle a lumen of FM4-64X–bound membrane dye. Single channel signals for MLCK, FM4-64X, and CTXB (left) show that FM4-64X signal is present in the ring, whereas CTXB signal is not. A three-dimensional reconstruction of the cell (right) with magnified panels adjacent shows that the MLCK ring surrounds FM4-64X dye. (D) Model for the visualization of rings. Side views of potential involuting membrane structures labeled with FM4-64X dye and recruited MLCK are marked with a plane of focus. The corresponding predicted two-dimensional patterns of FM4-64X and MLCK signal, as seen from above, are adjacent. Scale bars are 15 μm.