Fig. 5. Xenopus NEDD1 (XNEDD1) interacts with the TuRC in egg extracts. (A) Beads coated with recombinant GST, full-length GST-XNEDD1, GST-XNEDD1-N, GST-XNEDD1-C, or -tubulin antibodies were incubated with pure tubulin to assay their ability to nucleate microtubules. Beads were either used directly (left panels; not exposed to extract) or were first incubated in extract (middle panels, exposed to extract) or in -tubulin-depleted extract (right panels, exposed to extract but TuRC-). Representative pictures are shown for each condition. Beads were detected by autofluorescence; microtubules appear as spiky halos surrounding the beads. Scale bar, 10 µm. (B) Western blot of proteins that associate with the beads coated as in A and incubated in extract. The blot was probed with antibodies directed against the TuRC subunits Xgrip210, Xgrip109 or -tubulin, as indicated on the right. Protein `baits' are indicated above the lanes. Extr, extract control; GST, GST-coated beads; FL, full-length GST-XNEDD1; N, GST-XNEDD1-N; C, GST-XEDD1-C; IP, beads coated with antibodies against -tubulin. (C) Western blot of imunoprecipitations from egg extracts using antibodies directed against -tubulin or XNEDD1, as indicated above the blot. Input represents 1.5 µl of extract; immunoprecipitations were from 100 µl of extract.