Fig. 4. Xenopus NEDD1 (XNEDD1) depletion reduces the amount of -tubulin that associates with the centrosome but does not abolish -tubulin recruitment. (A,B) Micrographs of -tubulin staining at sperm heads incubated in buffer (top panels), mock-depleted extract (middle panels), or XNEDD1-depleted extract (bottom panels). Signals from the individual fluorescein (-tubulin, left column; green in overlay), rhodamine (-tubulin, center column; red in overlay), or UV (DNA stain; blue in overlay) are shown as overlays in the right column. Scale bar, 1 µm. (B) Quantification of the amount of -tubulin associated with sperm centrioles for the experiment shown in A, normalized against the -tubulin immunofluorescence for sperm tips incubated in mock-depleted extract. Error bars, SE. (C) Drosophila centrosomes were rendered inactive by treatment with KI, and were then incubated with mock-depleted (top row) or XNEDD1-depleted (bottom row) extract (see text for detail). The extract was then washed off and reconstituted centrosomes were incubated with a solution of pure tubulin (containing a small amount of rhodamine-tubulin) to assay their ability to nucleate microtubules. Microtubules were fixed, and visualized under the microscope. Centrosomes reconstituted with mitotic (left panels) or interphase (right panels) extract are shown. Scale bar, 5 µm. (D) Quantification of -tubulin recruited to reconstituted centrosomes treated as described in C, except that they were fixed and stained for -tubulin immunofluorescence instead of being tested in the microtubule nucleation assay. -tubulin immunofluorescence intensity is reported as percent of mock-depleted control. Error bars give the standard error (± s.e.). (E) Western blot showing the extent of XNEDD1 depletion (top panel) and lack of co-depletion of -tubulin (bottom panel). (F) Microtubule asters assembled in mock-depleted (top panel) or XNEDD1-depleted (bottom panel) extracts are stained for -tubulin (left row; green in overlay), -tubulin (second row; red in overlay) or DNA (third row; blue in overlay). (G) Quantification of -tubulin immunofluorescence relative to the amount of -tubulin fluorescence for the experiment shown in F. Error bars give the standard error (± s.e.).