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. 2016 Jan 15;27(2):295–307. doi: 10.1091/mbc.E15-08-0608

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© 2016 Harris 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 5: — Recovery of EB1-NG is not linked to tubulin exchange at the flagellar tip. (A–C) Gallery of individual frames (A), corresponding kymograms (B), and signal quantification (C) of a two-color FRAP experiment. (A) The distal flagellar region of a cell coexpressing EB1-NG and mCherry-α-tubulin was bleached using a laser spot that was moved along the flagellum (indicated by the dashed region), and FRAP was analyzed over several minutes (T0–T900 s). Top row, EB1-NG; bottom, mCherry-α-tubulin; arrowheads, flagellar tip. Scale bar: 1 μm. (B) Single-channel kymograms corresponding to A. The bleaching step and the orientation of the flagella are indicated. Scale bars: 1 μm and 10 s. (C) Quantification of the fluorescence intensity of EB1-NG (green) and mCherry-α-tubulin (red) corresponding to the photobleaching experiment depicted in A and B. The prebleach fluorescence intensity was set to 100 for both proteins; EB1-NG recovered rapidly and completely, while only traces of mCherry-α-tubulin were recovered even after 15 min of observation. See Supplemental Figure S4 for a similar experiment.