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Fig. 1. CAP350 localisation during the cell cycle and after microtubule drug treatment. (A) Western blot analysis of Triton X-100-soluble (S) and insoluble (I) protein fractions from KE37 cells and a highly enriched centrosomal fraction (CTR). CAP350 is only detectable in the centrosomal fraction. (B) Western Blot analysis of total cell lysate obtained from HeLa cells after CAP350 SiRNA. The high molecular mass band, revealed by CAP350 antibody, is detectable in the control siRNA sample (GL2 lane), and absent after CAP350 RNAi (O1 and O2 lanes) indicating that our antibody is specific. (C-F) Double-labelling experiments on interphase cells using the CAP350 antibody and either -tubulin (C-D), -tubulin (E) or CTR433 a Golgi complex antibody (F). Note that a cloud of dots is evident around the centrosome with the CAP350 antibody (arrow). (D) 3x magnification of the boxed area in C, showing that the pericentrosomal dots are aligned with MTs. These dots are located in the Golgi complex area (F). (G) Metaphase cell double stained with anti-CAP350 and anti--tubulin antibodies. During metaphase, CAP350 still localised to the centrosome, but was also found along the spindle microtubules. (H) Cell in telophase, double stained with anti-CAP350 and anti--tubulin antibodies. At this stage, the CAP350 antibodies labelled the centrosome but also some dots around it, as well as the central spindle. (I) Nocodazole (NZ)-treated cells double labelled with CAP350 and -tubulin antibodies during interphase. The CAP350 antibody decorated the centrosome and the remaining microtubules. (J) Taxol-treated cells double labelled with CAP350 and -tubulin antibodies. CAP350 redistributed to the ends of the microtubule bundles. Bars, 20 µm.

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